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な に ぬ

Modification of Gene Expression and Virulence ね Traits in Streptococcus mutans in Response to の

Carbohydrate Availability は ば

Zachary Moye, Lin Zeng, and Robert A. Burne* ぱ ひ

Department of Oral Biology, College of Dentistry, University of Florida, など Gainesville, Florida 32610 なな

なに Running Title: Response to feast and famine なぬ Key Words: Sugar transport, gene regulation, phosphotransferase system, dental caries, なね chemostat culture なの なは * Corresponding author なば Mailing address: なぱ

Department of Oral Biology, University of Florida, College of Dentistry, P.O. Box なひ 100424, Gainesville, FL 32610. にど Phone: (352) 273-8850 にな Fax: (352) 273-8829 にに E-mail: rburne@dental.ufl.edu にぬ にね

AEM Accepts, published online ahead of print on 22 November 2013Appl. Environ. Microbiol. doi:10.1128/AEM.03579-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract にの The genetic and phenotypic responses of Streptococcus mutans, an organism that is には strongly associated with the development of dental caries, to changes in carbohydrate にば availability were investigated. S. mutans UA159 or a derivative of UA159 lacking ManL, にぱ which is the EIIAB component (EIIABMan) of a mannose/glucose permease of the にひ phosphoenolpyruvate:sugar phosphotransferase system (PTS) and a dominant effector ぬど of catabolite repression, were grown in continuous culture to steady-state in conditions ぬな of excess (100 mM) or limiting (10 mM) glucose. Microarrays using RNA from S. mutans ぬに UA159 revealed that 174 genes were differentially expressed in response to changes in ぬぬ carbohydrate availability (P < 0.001). Glucose-limited cells possessed higher PTS ぬね activity, could acidify the environment more rapidly and to a greater extent, and ぬの produced more ManL protein than cultures grown with excess glucose. Loss of ManL ぬは adversely affected carbohydrate transport and acid tolerance. Comparison of the HPr ぬば protein in S. mutans UA159 and the manL deletion strain indicated that the differences in ぬぱ behaviors of the strains were not due to major differences in HPr pools or HPr ぬひ phosphorylation status. Therefore, carbohydrate availability alone can dramatically ねど influence the expression of physiologic and biochemical pathways that contribute directly ねな to the virulence of S. mutans, and ManL has a profound influence on this behavior. ねに ねぬ ねね ねの ねは ねば

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Introduction ねぱ Dental caries develops as a result of changes in the microbial composition and ねひ

biochemical activities of oral biofilms in response to alterations in environmental のど conditions; including nutrient source and availability, and repeated acidification (1). のな When the host is fasting, the oral flora catabolizes nutrients derived from host secretions のに and sloughed cells (1, 2). When the host consumes large quantities of sugary foodstuffs, のぬ multiple cariogenic species, including Streptococcus mutans, ferment these のね carbohydrates to organic acids that damage the tooth enamel (3-5). Analysis of the のの human oral microbiota in health and disease has provided evidence that a diverse のは community of organisms contributes to the initiation and progression of dental caries, のば although strong associations of elevated levels of S. mutans with the presence of active のぱ disease are consistently observed (3, 6). Dissection of the virulence of individual のひ organisms or combinations of oral bacteria has been complicated by the fact that はど phenotypic behaviors can be greatly impacted by environmental conditions (7). An はな understanding of how specific pathogens, such as S. mutans, cope with fluctuations in はに the chemical composition of the environment to compete with commensal members of はぬ the flora is essential to understanding the cariogenicity of the oral microbiome and for はね designing interventions to eliminate or reduce the proportions of cariogenic organisms in はの oral biofilms (8, 9). はは

S. mutans relies on the fermentation of dietary sugars to generate energy for はば growth, as it lacks a complete TCA cycle and respiratory chain (10). The organism はぱ expresses several pathways for the internalization of carbohydrates, including the はひ multiple-sugar metabolism (msm) ABC transport system that is capable of internalizing ばど raffinose, melibiose and isomaltosaccharides (11). However, most of the carbohydrates ばな catabolized by S. mutans are internalized by the phosphoenolpyruvate:sugar ばに

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phosphotransferase system (PTS) (12). The general PTS proteins Enzyme I (EI) and ばぬ Histidine Protein (HPr) participate in the transport of all PTS sugars. A variety of sugar-ばね specific Enzyme II (EII) permeases are commonly present in S. mutans (10, 13-15), and ばの are composed of cytoplasmically-located A and B subunits, as well as C and in some ばは cases D domains embedded in the cell membrane (12). More than fourteen unique PTS ばば permeases that transport a spectrum of mono- and di-saccharides, including glucose, ばぱ sucrose, mannose, fructose, cellobiose, lactose and maltose, are present in the ばひ reference strain S. mutans UA159 (13, 15). Rapid fermentation of sugars by S. mutans ぱど can cause a substantial drop in the pH of oral biofilms, from neutral pH to values well ぱな below 5 (16). The acidic environment created by carbohydrate fermentation favors the ぱに growth of S. mutans over health-associated, acid-sensitive commensal organisms, ぱぬ because S. mutans is well adapted to survive and continue to metabolize carbohydrates ぱね at low pH (17). ぱの

In low G+C Gram-positive bacteria, the regulation of carbohydrate catabolite ぱは repression (CCR), the ability to selectively catabolize preferred carbohydrates when non-ぱば preferred carbohydrates are also present, is typically controlled by HPr of the PTS and ぱぱ the LacI/GalR-type catabolite control protein A (CcpA) (18). Interestingly, it has been ぱひ demonstrated that CcpA has very little influence on CCR in S. mutans (19-21), whereas ひど transcriptomic analyses have revealed a prominent role for CcpA in regulation of global ひな carbon flow; controlling the transition between homo- and hetero-fermentative growth ひに and regeneration of NADH, as failure to maintain adequate NADH levels in S. mutans ひぬ can have detrimental effects (14, 22). While it has been shown that HPr can exert effects ひね on CCR in S. mutans, CCR in this organism is dominantly controlled by the ひの mannose/glucose EIIAB permease, ManL (EIIABMan), but also the FruI and FruCD ひは fructose permeases, the EIILev fructose/mannose permease and sucrose PTS permease ひば

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have been shown to be capable of influencing CCR in this cariogenic pathogen when ひぱ their cognate sugars are present (23, 24). Notably, deletion of manL resulted in a loss of ひひ diauxic growth in medium containing a combination of glucose and the fructose などど homopolymer inulin as the carbohydrate sources, and a general disregulation of many などな genes involved in carbohydrate transport, catabolite repression and energy metabolism などに was noted in a manL deletion mutant when compared with the parental strain, UA159 などぬ (25). Thus, the current working model for CCR in S. mutans posits that information on などね carbohydrate flow through the glycolytic pathway is transduced by phosphorylated などの derivatives of HPr, while PTS permeases monitor the source and availability of specific などは carbohydrates in the environment (26). などば

The use of continuous culture has proven to be a powerful tool for dissecting the などぱ impact of carbohydrate availability, growth rate and pH on the physiology of S. mutans, などひ although most of the studies were done when technologies were not available to ななど correlate the results with gene expression profiles (27-32). For example, when cultures ななな of S. mutans strain Ingbritt were grown to steady state with excess glucose, the cells ななに displayed a slight drop in glycolytic rate when cultured at lower pH or faster growth rates. ななぬ A similar pattern was observed for strain Ingbritt grown under glucose limitation, ななね although the decrease in glycolytic rate was more pronounced (27, 30). The glycolytic ななの rate for glucose, fructose and sucrose was higher in cells of S. mutans Ingbritt grown ななは under glucose limitation versus glucose excess, whereas the rate of glycolysis of ななば endogenous stores of carbohydrates was higher in cells grown with excess glucose. ななぱ Likewise, cells limited for carbohydrate were able to lower the pH faster using ななひ exogenous sources, while cells grown in excess glucose contained greater endogenous なにど stores of carbohydrates (28). When sugar transport was assayed in S. mutans Ingbritt なにな and the clinical isolate 123.1, PTS-dependent transport of glucose, mannose and 2-なにに

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deoxyglucose was lower when cells were grown at higher dilution rates, lower pH or with なにぬ excess glucose (27, 29, 31); although fructose PTS activity did not follow the same trend なにね (31). A decrease in EII activity was noted as strain Ingbritt or strain 123.1 was grown in なにの increasing concentrations of glucose (31, 32). Similarly, EI protein levels were found to なには be lower in glucose excess conditions, compared to glucose limitation, except when cells なにば where grown at a very low growth rate at pH 7 (31, 32). In S. mutans Ingbritt, the amount なにぱ of HPr was shown to decrease slightly with increasing concentrations of glucose (32), なにひ whereas others have reported that the levels of HPr in S. mutans 123.1 remain relatively なぬど constant (31). Multiple studies demonstrated that PTS-dependent transport did not なぬな always correlate with the total growth rate of cells, and this effect was more pronounced なぬに for cells grown at high growth rates (27, 33, 34). なぬぬ

Clearly, the availability of carbohydrates has substantial effects on the physiology なぬね and pathogenic potential of S mutans, and in a few instances, these adaptations have なぬの begun to be understood at the biochemical level. However, the genetic regulatory なぬは circuits underlying these adaptations are only beginning to be appreciated, and a limited なぬば understanding of the scope and complexity of the responses is presently available. The なぬぱ goals of this study were to more fully characterize the mechanisms utilized for adaptation なぬひ to carbohydrate availability by S. mutans in the context of the major regulator of CCR, なねど ManL, and to begin to integrate genetic data with physiologic behaviors. The results なねな provide new insights into how this dental pathogen alters its virulence potential in なねに response to the major driver of dental caries, carbohydrate availability. なねぬ なねね なねの なねは

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Materials and Methods なねば Bacterial strains and growth conditions. Streptococcus mutans strain UA159 was なねぱ maintained using brain-heart infusion (BHI) medium and routinely cultured in a tryptone-なねひ vitamin (TV) base medium supplemented with glucose (35). For continuous culture なのど studies, cells were cultivated in a Biostat i® Twin chemostat system (B. Braun Biotech, なのな Inc., Allentown, PA) with a working volume of 0.5 L. Cultures were grown in TV base なのに medium supplemented with either 10 mM (limiting) or 100 mM (excess) glucose (35). In なのぬ all cases, concentrated glucose solutions were briefly autoclaved or filter-sterilized, なのね combined with a filter-sterilized vitamin mixture and aseptically added to a vessel なのの containing sterile tryptone base medium. To begin each experiment, a chemostat vessel なのは containing 500 ml of sterile TV medium with 0.5% glucose was inoculated with 5 ml of an なのば overnight culture of S. mutans grown in BHI broth. Cells were grown to OD600 = 0.7, at なのぱ which time medium was pumped into the vessel at a dilution rate (D) equal to 0.3 h-1; なのひ corresponding to a generation time of 2.3 h at steady-state (36). Steady state was なはど assumed to have been reached after 10 generations (36). The availability of glucose なはな was monitored by measuring free glucose in the culture supernatant fluid using a なはに glucose oxidase assay (Sigma, St. Louis, MO) with a lower limit of detection of なはぬ approximately 1 µg ml-1. The pH of the medium was maintained at 7.0 by the addition of なはね 1 M KOH, the temperature was kept at 37°C and the culture was stirred at 200 RPM. なはの Once steady state was established, 250 ml of the cell culture was removed from the なはは vessel and immediately chilled on ice. Cells were quickly aliquoted into smaller volumes なはば and centrifuged at 4°C. In some instances, cells were resuspended and utilized なはぱ immediately in assays, while others were snap frozen in a dry ice-ethanol bath and なはひ stored at -80°C. In other cases, pellets were treated with RNAprotect Bacteria Reagent なばど (Qiagen, Inc, Chatsworth, CA), snap frozen and stored at -80°C to preserve RNA for なばな

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microarray and qRT-PCR studies. Three replicates of cultures grown in 10 mM or 100 なばに mM glucose were generated. なばぬ pH drop assays. Bacterial cultures were subjected to pH drop experiments as なばね previously described (22, 37). Briefly, samples of the cultures were collected and なばの centrifuged at 4ºC. Cells were resuspended in cold deionized water and the OD600 of the なばは suspension was adjusted to 0.5. Aliquots (50 ml) of the normalized cell suspensions なばば were then centrifuged at 4°C and resuspended in 4.75 ml of 50 mM KCl, 1 mM MgCl2. A なばぱ small stir bar was added and the pH of the cell suspension was adjusted to 7.2 by the なばひ addition of 0.1 M KOH. Once a stable pH was established, 0.25 ml of a 1 M glucose なぱど solution was added and the decrease in the pH was monitored continuously for 30 なぱな minutes using a pH meter connected to a computer. For assays of acid production from なぱに endogenous sources, cells were harvested and resuspended in cold deionized water, なぱぬ and the OD600 values of the cell suspensions were adjusted with cold water to 0.5. Cells なぱね were collected as above, resuspended in 5 ml of 50 mM KCl, 1 mM MgCl2, the pH of the なぱの suspension was rapidly adjusted to pH 7.2 with KOH, and the pH of the cell suspension なぱは was monitored over time without addition of glucose. なぱば PTS assays. The ability of cells to transport sugars by the PTS was assessed as なぱぱ described elsewhere (38, 39), with minor modifications. Briefly, frozen cell pellets were なぱひ thawed on ice and the OD600 of the suspensions was adjusted to 0.5. The cells were なひど washed twice with 0.1 M sodium-potassium phosphate buffer and resuspended in 0.1 なひな volume of the same buffer. Previous analyses conducted in our laboratory showed that なひに snap freezing and storage at -80°C does not affect PTS activity. Cells were なひぬ permeabilized by addition of 0.05 volumes of a toluene:acetone (1:9, v/v) solution and なひね vortexing for two minutes, twice. Reactions included permeabilized cells, 100 µM NADH, なひの 10 µM NaF, 10 µM of the desired carbohydrate and 10 µl of a lactate dehydrogenase なひは

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solution (13.1 kunits/ml Sigma, St. Louis, MO) in 0.1 M sodium potassium phosphate なひば buffer. The reaction was conducted at 37°C and initiated by the addition of 5 mM なひぱ phosphoenolpyruvate (PEP). The rate of PEP-dependent oxidation of NADH was なひひ monitored over time and used to calculate reaction rates, which were then normalized to にどど the concentration of protein measured using a bicinchoninic acid (BCA) assay (Thermo にどな Scientific, Rockford, IL) with bovine serum albumin as the standard. にどに Immunoblot assays. A polyclonal antibody was raised against a purified, recombinant にどぬ ManL protein. To express ManL, oligonucleotide primers were synthesized to PCR-にどね amplify the manL coding sequence and to introduce 5’ BamHI and 3’ HindIII restriction にどの sites. The resulting PCR product was digested with BamHI and HindIII and ligated into にどは plasmid pQE-30, encoding a 6-histidine N-terminal tag in frame with the manL gene. The にどば ligation mixture was transformed into Escherichia coli strain M15. DNA sequencing was にどぱ used to verify that the fusion protein was intact. The recombinant strain was grown in 1 L にどひ of L broth to OD600 of 0.5, IPTG was then added to a final concentration of 1 mM and the になど culture was incubated for an additional 4 hours. Following purification by metal affinity になな chromatography, approximately 1 mg of purified, recombinant ManL was になに electrophoresed in an SDS-PAG. Slices of the gel containing ManL were sent to Lampire になぬ Biologicals (Pipersville, PA), where a rabbit polyclonal antiserum was generated. になね Antisera generated against the EI and HPr proteins from S. mutans strain DR0001 (40) になの were a kind gift from Christian Vadeboncouer. になは

S. mutans cell lysates were generated in a Bead Beater (Biospec, Bartlesville, になば OK) using 0.5 ml of glass beads (average diameter, 0.1 mm) in 750 µl of 1X PBS, the になぱ lysates were clarified by centrifugation at 16,000 x g at 4°C for 10 minutes and the になひ protein concentration of clarified cell lysates was measured using a commercially-ににど available Bradford assay (BioRad, Hercules, CA). Equal amounts of protein from each ににな

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cell lysate were boiled for 5 minutes in SDS sample buffer, separated by SDS-PAGE ににに and transferred to a PVDF membrane using a Trans-Blot SD (BioRad). Membranes ににぬ were blocked with Tris-buffered saline (10 mM Tris-HCl, pH 7.4, 0.9% NaCl, 0.05% ににね Tween-20) containing 5% non-fat dried milk, then incubated with primary antisera, ににの washed and incubated with a goat anti-rabbit peroxidase-conjugated antibody (1:4000 にには dilution; KPL, Gaithersburg, MD). After additional washing, the membranes were ににば developed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo ににぱ Scientific, Rockford, IL). The different species of HPr (26) were detected in cell lysates ににひ that had either been kept on ice or boiled to remove the phosphate moiety from にぬど Histidine-15. After separation of the proteins by non-denaturing PAGE and transfer to a にぬな PVDF membrane, HPr was detected by immunoblotting using the polyclonal anti-HPr にぬに antiserum described above. Quantification of signals was performed using densitometry. にぬぬ RNA isolation. RNA was extracted from cells as described previously, with some にぬね modifications (22). Briefly, 50 ml of cells from the chemostat were pelleted, resuspended にぬの in one ml of RNAprotect Bacteria Reagent and incubated at room temperature for 10 にぬは minutes. The cell suspensions were then pelleted, the supernatant fluid was removed, にぬば cell pellets were quickly frozen using dry ice and stored at -80°C. Frozen cells were にぬぱ thawed on ice, resuspended in 5 ml of 50:10 TE buffer (50 mM Tris:10 mM EDTA) and にぬひ diluted to equal cell densities. Then, 250 µl of each cell suspension was mixed with 300 にねど µl of acidic phenol and placed in 1.5 ml screw cap tubes with 0.2 ml of glass beads にねな (average diameter, 0.1 mm). Cells were homogenized by Bead-beating for 30 seconds, にねに two times, with placement on ice for 2 minutes between cycles. The lysates were にねぬ centrifuged for 10 minutes, 150 µl of the aqueous phase was removed and RNA was にねね extracted using the Qiagen RNeasy extraction kit (Qiagen, Inc). Extracts were then にねの subjected to two separate treatments with an RNase-free DNase I (Qiagen, Inc). RNA にねは

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was transferred to an RNase-free column, washed and eluted in 30 µl of RNase free にねば water. にねぱ Microarray experiments. Microarray slides containing 1,948 70-mer oligonucleotide にねひ open reading frames of S. mutans UA159 printed 5 times on glass slides were obtained にのど from the Pathogen Functional Genomics Resource Center (PFGRC) にのな (http://pfgrc.jcvi.org) (22). In order to account for biases arising from array data due to にのに differences in the fluorescent signal intensity of the two dyes used, reference RNA was にのぬ prepared from a 50 ml culture of S. mutans UA159 grown to mid-exponential phase in にのね BHI broth (22). RNA that was purified as described above was used to generate にのの aminoallyl-labeled cDNA using protocols provided by the PFGRC にのは (http://pfgrc.tigr.org/protocols.shtml). Briefly, two µg of RNA from each of the three にのば replicates from chemostat cells and samples of reference RNA were used to generate にのぱ cDNA by amplification with random hexamer primers using Super-Script III reverse にのひ transcriptase (Invitrogen, Gaithersburg, MD). To label the cDNA, aminoallyl-dUTP (aa-にはど dUTP) (Sigma) was added to the nucleotide pool during the reverse transcription にはな reaction at a ratio of 2:1 to dTTP. The fluorescent molecule indocarbocyanine Cy3 was にはに used to label the experimental group cDNA while indocarbocyanine Cy5 (Amersham にはぬ Biosciences, Piscataway, NJ) was coupled to the reference group cDNA. Based on the にはね measured dye incorporation, experimental group cDNA was mixed with reference cDNA にはの possessing similar dye incorporation and hybridized to prepared slides using a Maui にはは four-chamber hybridization system (BioMicro Systems, Salt Lake City, UT) for 16 h at にはば 42°C. The slides were washed according to the PFGRC protocols and prepared for にはぱ scanning using a GenePix scanner (Axon Instruments Inc., Union City, CA). にはひ

Scanned slides were analyzed using the PFGRC Spotfinder software にばど (http://www.tm4.org/spotfinder.html) and Cy3 and Cy5 images were overlaid. Using the にばな

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software, a spot-locating grid was created and used to identify spots and determine spot にばに intensity. The Microarray Data Analysis Software (MIDAS) from the PFGRC was used to にばぬ normalize data collected from the overlaid spot grids (http://www.tm4.org/midas.html). にばね BRB Array Tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) was used to generate the にばの statistical data with a p-value of 0.001 as the cut-off. にばは Real-time quantitative RT-PCR. RNA was isolated from experimental samples as にばば described above. Using one µg of RNA, Super-Script III reverse transcriptase was にばぱ utilized to generate cDNA using gene specific antisense primers. Gene specific PCR にばひ products were created and used as standards. The preparation of standards and data にぱど analysis were conducted as described elsewhere (25). にぱな Microarray data accession number. Microarray data has been deposited at NCBI-にぱに GEO (GSE51362). にぱぬ

にぱね Results にぱの

Acid production. The ability of S. mutans strains to lower the pH when provided with にぱは excess exogenous glucose was monitored by a pH drop assay, as detailed in the にぱば Methods section. Cell suspensions of S. mutans UA159 that had been cultured to steady にぱぱ state in glucose-limiting (10 mM glucose) conditions rapidly lowered the pH to an にぱひ average value after 30 minutes of 3.50 ± 0.08 (Table 1). In contrast, wild-type cells that にひど had been grown with excess glucose (100 mM glucose) lowered the pH to an average of にひな 3.70 ± 0.01 during the same time interval (Figure 1A; P = 0.047). Moreover, glucose-にひに limited cells were able to lower the pH at a faster rate, as illustrated by the fact that the にひぬ pH of cell suspensions fell to an average of 5.39 ± 0.31 after 2 minutes, whereas wild-にひね type cells grown in excess glucose reached a pH of 6.23 ± 0.08 in the same time period にひの

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(Figure 1A; P = 0.035). It was also noted that cells grown in excess glucose required にひは substantially more KOH to equilibrate the pH before beginning the assay than glucose-にひば limited cells, presumably due to metabolism of endogenous stores of glycogen-like にひぱ polysaccharides. To confirm this, we rapidly neutralized fresh cell suspensions to a pH of にひひ 7.2 and monitored the pH for 30 minutes without adding exogenous glucose. These ぬどど results demonstrated that S. mutans UA159 grown with excess glucose lowered the pH ぬどな to an average of 6.39 ± 0.20, whereas cells grown in glucose-limiting conditions lowered ぬどに the pH to 6.68 ± 0.06 (Figure 1B; P = 0.119). ぬどぬ Interestingly, cell suspensions of the manL mutant cultures that had been grown ぬどね to steady state in limiting glucose were able to lower the pH after 30 minutes to an ぬどの average of 3.88 ± 0.12, whereas cells grown in excess glucose lowered the pH to an ぬどは average of 3.76 ± 0.05 (Figure 1C; P = 0.220). Additionally, glucose-limited manL ぬどば mutant cells consistently lowered the pH at a faster rate (6.03 ± 0.21 after 2 minutes) ぬどぱ than cells grown in excess glucose (6.30 ± 0.20 in the same time period); although the ぬどひ differences were not statistically significant between glucose-excess and -limited cells (P ぬなど = 0.195). As mentioned above, glucose-limited wild-type cells were able to lower the pH ぬなな to an average of 5.39 ± 0.31 after 2 minutes, and this was significantly different from the ぬなに manL mutant grown in limiting glucose (P = 0.047) or excess glucose (P = 0.018); ぬなぬ although there were no significant differences in the pH values attained by the wild-type ぬなね and manL mutant cell suspensions when grown in excess glucose (P = 0.616). Thus, the ぬなの loss of ManL dramatically impacts glycolytic rates when cells are grown in limiting ぬなは glucose. Also of note, the terminal pH achieved in the pH drop assay is strongly ぬなば correlated with acid tolerance (17, 41, 42); lower pH being associated with greater acid ぬなぱ tolerance. The fact that cells that lacked ManL and were limited for glucose could not ぬなひ achieve as low a terminal pH as the wild-type strain grown under the same conditions ぬにど

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implicates ManL as a potential contributor to, or regulator of, acid tolerance. We also ぬにな attempted to quantify the acid tolerance of cells using a typical acid killing assay (37). ぬにに However, exposure to pH 2.8 of fresh chemostat cultures of both the wild-type and manL ぬにぬ deletion strain grown with excess glucose caused the cells to aggregate into large ぬにね clumps very rapidly. Despite multiple attempts to disperse the cells, it was not possible ぬにの to reliably enumerate CFU in these populations of cells following an acid challenge. ぬには Initial investigation into the cause of the clumping at low pH suggested it was, at least in ぬにば part, associated with enhanced cell surface hydrophobicity (data not shown). ぬにぱ PTS activity. To test whether the enhanced generation of acid by glucose-limited cells ぬにひ could be associated with more efficient internalization of carbohydrates by the PTS, ぬぬど PTS-dependent transport of carbohydrates by cells grown under conditions of glucose ぬぬな limitation or excess was compared. The transport of mannose, fructose or glucose by ぬぬに the PTS was markedly higher in wild-type cells grown under conditions of glucose ぬぬぬ limitation than in cells grown with excess glucose (Figure 2A). This finding is consistent ぬぬね with, and may in large part explain, the faster rate at which cells grown under conditions ぬぬの of limiting glucose can lower the pH (Figure 1A). Conversely, the manL mutant strain ぬぬは displayed very low levels of PTS-dependent transport of glucose and mannose. These ぬぬば results agree with the pH drop experiments in that the manL mutant strain showed a ぬぬぱ consistently lower rate and extent of acidification when grown under glucose limitation. ぬぬひ Notably, transport of fructose by cells grown under glucose limitation was also lower for ぬねど cells lacking ManL than for the wild-type strain, but no difference in PTS-dependent ぬねな fructose transport was observed between wild-type and manL mutant cells grown with ぬねに excess glucose. ぬねぬ

To determine if the effects on PTS-dependent carbohydrate transport in the ぬねね manL mutant were associated with a general defect in the PTS or were associated only ぬねの

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with loss of ManL, which has been shown to transport glucose, mannose and galactose, ぬねは PTS-dependent transport assays were performed using maltose, cellobiose or lactose ぬねば as the substrate. For all three disaccharides, cells grown with glucose limitation were ぬねぱ more efficient at transporting these carbohydrates than cells grown in glucose excess ぬねひ (Figure 2B). Cells bearing a deletion in manL displayed reduced capacity for PTS-ぬのど dependent transport of cellobiose and maltose (Figure 2B). PTS-dependent transport of ぬのな lactose was generally lower than the other sugars tested, probably due to the ぬのに requirement for lactose in the growth medium to induce the lac operon in S. mutans (43, ぬのぬ 44). ぬのね PTS enzyme levels and carbohydrate availability. To probe in more detail the basis ぬのの for the decreased PTS-dependent carbohydrate transport observed in cells grown in ぬのは excess glucose, immunoblotting was used to compare the levels of selected PTS ぬのば proteins in different growth conditions and strains. To perform this analysis, we ぬのぱ generated antisera in a rabbit against a full-length, histidine-tagged S. mutans ManL ぬのひ protein that was expressed in E. coli, purified by nickel affinity chromatography, and ぬはど further purified by SDS-PAGE and excision of gel slices. Immunoblotting confirmed that ぬはな the antibody specifically recognized a protein band with the predicted size of ManL (~35 ぬはに kDa) in batch-grown cells cultured in glucose or fructose, and that this band was absent ぬはぬ in cells carrying a non-polar deletion:replacement of the manL gene (Figure S1)(45). We ぬはね next probed cell lysates of S. mutans UA159 from glucose-limited and glucose-excess ぬはの steady-state cultures and consistently found more ManL protein in glucose-limited cells ぬはは than in cells grown with excess glucose (Figure 3A). Immunoblotting of the strain bearing ぬはば a manL deletion grown in continuous culture in limiting or excess glucose was performed ぬはぱ and revealed no protein bands under either condition (data not shown). ぬはひ

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In contrast to the results with ManL, when immunoblots were performed using ぬばど antibodies against the general PTS proteins, EI and HPr, very little change was evident ぬばな in the amount of these proteins in the wild-type cells in glucose-limiting and -excess ぬばに conditions (Figure 3A). Similar results were seen for the amounts of EI and HPr protein ぬばぬ in the manL deletion strain (Figure 3B). Immunoblot analysis was performed using whole ぬばね cell lysates of at least three separate replicates and, by using densitometry, it was ぬばの determined that the level of EI and HPr protein never differed by greater than 40% ぬばは between cells grown in limiting or excess glucose. We also performed qRT-PCR to ぬばば compare the expression levels of manL, ptsI (EI) and ptsH (HPr) using RNA generated ぬばぱ from chemostat samples of wild-type cultures. We observed consistently lower levels of ぬばひ manL mRNA by Real-Time PCR in cells grown with excess glucose (data not shown) in ぬぱど agreement with our microarray results (Table 3). In contrast, the transcript levels of EI ぬぱな (ptsI) and HPr (ptsH) did not differ between the two wild-type cell populations (Figure S2). ぬぱに

Our previous studies with S. mutans UA159 have provided evidence that HPr ぬぱぬ phosphorylated on serine 46 (HPr-Ser-P) can regulate carbohydrate transport (26). To ぬぱね probe whether the phosphorylation state of HPr was altered by the deletion of manL and ぬぱの could therefore be a potential contributor to the observed decrease in overall PTS ぬぱは activity, we subjected cell lysates to non-denaturing PAGE and performed ぬぱば immunoblotting with HPr antiserum. The wild-type and manL mutant strains grown under ぬぱぱ conditions of glucose excess displayed prominent bands corresponding to HPr(Ser-P) ぬぱひ and HPr(Ser-P)(His~P), whereas cells grown under glucose limitation completely lacked ぬひど these bands and contained mostly HPr(His~P) (Figure 4). Although we consistently ぬひな observed slightly lower levels of the serine-phosphorylated forms of HPr in the manL ぬひに mutant, HPr(Ser-P) and HPr(Ser-P)(His~P) were nearly as abundant and readily ぬひぬ detected in the manL mutant grown in glucose excess conditions. ぬひね

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The transcriptome of Streptococcus mutans UA159 is altered in response to ぬひの carbohydrate availability. The transcriptional changes present in steady-state ぬひは planktonic cultures of S. mutans UA159 were monitored as a function of glucose ぬひば availability. Comparison of the transcriptome of cells grown in limiting versus excess ぬひぱ glucose revealed that 174 genes were differentially expressed (P < 0.001), with 81 ぬひひ transcripts up-regulated and 93 down-regulated under conditions of limiting glucose, ねどど when compared to excess glucose growth conditions (Table 2). The validity of the ねどな microarray results was confirmed by subjecting a subset of genes to Real-Time qPCR ねどに (Table 3). ねどぬ Cells grown in limiting carbohydrate showed higher expression of genes ねどね associated with carbohydrate transport and energy metabolism, suggestive of a broad ねどの relief of catabolite repression. Interestingly, we observed that transcripts of the EII ねどは enzyme for fructose (SMU.115) and the co-transcribed fructose-1-phosphate kinase, a ねどば second fructose PTS operon including the EII enzyme (SMU.872), an associated ねどぱ transcriptional repressor and a second fructose-1-phosphate kinase, the predicted ねどひ ribulose-monophosphate PTS EII enzyme and an associated metabolic gene encoding ねなど hexulose-6-phosphate isomerase, as well as glucose/mannose and trehalose PTS EII ねなな permeases were elevated. Also of note, the gene for the transcriptional regulator CcpA ねなに was more highly expressed in glucose-limited cells. Again related to sugar metabolism, ねなぬ we noted an elevated level of transcripts encoding ScrB, sucrose-6-phosphate hydrolase ねなね and several transcripts of the msm ABC transporter system, including msmE, msmF, ねなの msmG, gtfA, msmK and dexB. Collectively, these results provide evidence that glucose-ねなは limited cells adjust gene expression to optimize the capacity for metabolism of other ねなば carbohydrates, even in the absence of inducing sugars. ねなぱ

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One of the most striking differences between conditions of limiting and excess ねなひ glucose was found in the category of energy metabolism. Under conditions of limiting ねにど glucose, a transcript of the Leloir pathway (galT) and a galactose-6-phosphate ねにな isomerase (lacB) of the tagatose pathway for galactose metabolism were elevated. ねにに Notably, the genes for metabolism of glycogen, glycogen phosphorylase (phsG), ねにぬ glycogen synthase (glgA) and other glycogen metabolic genes (glgB, glgC, glgD) were ねにね upregulated. Two genes encoding components of the pyruvate dehydrogenase (PDH) ねにの complex, SMU.1422 and SMU.1423, were also upregulated in glucose-limited cells and ねには represented the most highly upregulated genes. Also related to energy metabolism, ねにば transcripts for a putative phosphoglucomutase (pgm), adhE encoding a putative ねにぱ alcohol/acetaldehyde dehydrogenase, the water-forming NADH oxidase, pyruvate ねにひ formate-lyase and a putative succinate semialdehyde dehydrogenase were elevated ねぬど under conditions of glucose limitation. Finally, we noted the upregulation of several ねぬな genes related to cell processes that were of interest, including the gene for the molecular ねぬに chaperone DnaK and SMU.1425 (clpB), which encodes the ATP-binding subunit of a ねぬぬ putative Clp proteinase. ねぬね Cells grown under conditions of excess carbohydrate displayed a very different ねぬの transcriptome than those cultured in limiting glucose. Of particular interest, we noted the ねぬは upregulation of the transcriptional regulator codY and the associated transcript pncA. ねぬば Genes for two putative amino acid permeases (SMU.1450 and SMU.951), a subunit ねぬぱ (OpuCa) of the putative osmoprotectant amino acid ABC transporter and a subunit of an ねぬひ unrelated putative amino acid ABC transporter were upregulated. We also noted the ねねど transcription of a predicted zinc-dependent protease, SMU.1784c, which encodes a ねねな potential homolog of the Enterococcus faecalis membrane associated protease Eep (46), ねねに as well as two proteases resembling collagenase, SMU.759 and SMU.761, were ねねぬ

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elevated under glucose excess conditions. In addition, we observed the upregulation of ねねね several genes related to amino acid biosynthesis, including threonine dehydratase, ねねの glutamine synthetase, threonine synthase, argininosuccinate lyase and a putative ねねは NADP-specific glutamate dehydrogenase. The increased expression of this group of ねねば genes may signal the activation of amino acid scavenging pathways, consistent with the ねねぱ idea that cells growing with excess glucose are likely limited for an amino acid or group ねねひ of amino acids (27, 28). Finally, related to signals of depleted resources, we noted ねのど elevated transcription of phoH, which encodes a protein associated with phosphate ねのな starvation. ねのに Our microarray results also revealed an upregulation of genes instrumental in ねのぬ transport of solutes across the membrane, biosynthesis of nucleotide precursors and ねのね components needed for cell wall integrity in cells grown in excess glucose. Genes ねのの related to transport included a glycerol uptake facilitator permease, the ammonium ねのは transporter NrgA, a putative multidrug efflux pump, a putative sodium-dependent ねのば transporter and a subunit of an ABC transporter of unknown function (SMU.1315c). With ねのぱ regard to metabolism of nucleotides and nucleotide precursors, a number of genes ねのひ displayed elevated transcription, including guaB involved in GTP biosynthesis, pyrD for ねはど pyrimidine biosynthesis, purL and purC for purine metabolism, add for adenosine ねはな metabolism and kitH, encoding a putative thymidine kinase. Also of interest, transcripts ねはに for several cell wall biosynthesis gene products, including undecaprenyl pyrophosphate ねはぬ synthetase, UDP-N-acetylmuramyl tripeptide synthetase, UDP-N-acetylglucosamine 2-ねはね epimerase, a putative peptidoglycan hydrolase and a putative glycosyltransferase (csbB) ねはの involved in cell stress were elevated under glucose excess conditions. Finally, a few ねはは genes related to the metabolism of cofactors were upregulated, the most notable among ねはば these genes were a putative biotin biosynthesis protein (SMU.1827) and thiD, which is ねはぱ

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involved in thiamine metabolism, as well as other genes associated with energy ねはひ metabolism, including gapN (SMU.676) and capP (SMU.712) and a putative protein ねばど maturation peptidase precursor (prtM). ねばな

Discussion ねばに The intermittent introduction of, and variety in the types of, carbohydrates in the ねばぬ

diet of the human host presents multiple challenges to members of the oral microbiota, ねばね including S. mutans (7, 9). Of particular relevance to this study is that a delay in the ねばの induction of expression of transporters and catabolic pathways for particular ねばは carbohydrates that are consumed by the host could render S. mutans less competitive ねばば with commensal bacteria and impede its ability to contribute to the caries process. In our ねばぱ study, cells grown under glucose limitation displayed a general trend of increased PTS-ねばひ dependent transport efficiency across all sugars tested (Figure 2). These results agree ねぱど with and significantly extend previous studies that showed enhanced transport of ねぱな glucose, mannose and 2-deoxyglucose in carbohydrate-limited cells; although glucose ねぱに concentrations, dilution rates and the strain utilized in our study differed from those in ねぱぬ previous studies (31, 32). ねぱね

The diminished level of PTS-dependent sugar transport detected in both the wild-ねぱの type and manL deletion strains when cells were grown with excess glucose could be ねぱは related to accumulation of HPr(Ser-P) and the dually-phosphorylated form of HPr, ねぱば HPr(Ser-P)(His~P) (Figure 4). These observations are consistent with a previous report ねぱぱ where an increase in HPr(Ser-P)(His~P) and HPr(Ser-P) species was evident when S. ねぱひ mutans strain Ingbritt was grown in continuous culture in medium containing excess ねひど glucose (47). HPr is a central effector of catabolite repression and its phosphorylation ねひな state can have a significant impact on growth and carbohydrate transport, as well as on ねひに

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expression of virulence-related genes by enhancing the binding of CcpA to catabolite ねひぬ responsive elements (CREs) (18, 26, 48). We suggest that the repression of PTS-ねひね dependent transport noted when cells were grown with excess glucose could be ねひの attributed in part to interference of HPr(Ser-P) with carbohydrate transporters, which ねひは would prevent the internalization of the inducing sugar; a phenomenon known as inducer ねひば exclusion (48). Evidence for HPr-dependent interference with carbohydrate transport has ねひぱ been observed in genetically-modified derivatives of S. mutans UA159 expressing ねひひ elevated levels of HPr(Ser-P) or an HPr(Ser-P) mimic, the latter being an HPr protein のどど carrying an aspartic acid residue in place of serine 46. Strains carrying this serine to のどな aspartic acid mutation displayed repression of PTS-dependent uptake of glucose, のどに fructose and mannose, as well as diminished growth on lactose and cellobiose (26). のどぬ Thus, HPr phosphorylation status, perhaps coupled with changes in ccpA expression, のどね must be considered as a possible explanation for the phenotypic and transcriptomic のどの differences in cells cultured in limiting versus excess glucose. In contrast, the fact that のどは the pools of modified and unmodified HPr in the wild-type and manL mutant strains were のどば generally similar (Figure 4) supports the notion that differences in the phenotypic のどぱ behaviors of the wild-type and manL mutant strain arise primarily from loss of ManL, のどひ rather than from major changes in the amount or phosphorylation status of HPr. のなど

Previous studies have demonstrated that cells of S. mutans grown with excess のなな glucose in continuous culture conditions were limited for aspartate and asparagine at のなに high dilution rates, whereas only cysteine was found to be limiting at low dilution rates のなぬ (27-29). Our microarray data revealed elevated transcription of genes related to the のなね transport of amino acids and amino acid biosynthesis when cells were grown in excess のなの glucose. Further, we noted the upregulation of the transcriptional regulator CodY, which のなは is a global regulator of gene expression that responds to the levels of branched-chain のなば

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amino acids. These data provide strong evidence that the organism is indeed limited for のなぱ amino acids during periods of growth with excess carbohydrate. Several of the genes のなひ that were upregulated when cells were grown with excess glucose were annotated as のにど amino acid or ion transporters, as well as multiple uncharacterized transporters. Given のにな the potentially important role that these transporters play under conditions that are のにに conducive to the development of caries, i.e. when excess carbohydrates are available, のにぬ future studies identifying ways to inhibit the acquisition of key nutrients by S. mutans のにね during carbohydrate excess may discourage the emergence of this caries pathogen in のにの oral biofilms. のには

Previous investigators have shown that there is insufficient PTS-dependent のにば transport in cells growing with excess carbohydrate to account for the total amount of のにぱ glucose internalized, and it has been suggested that an alternative glucose uptake のにひ system must exist (27, 33, 49, 50). The most convincing evidence for this theory is that a のぬど ptsI mutant strain, lacking Enzyme I, was able to grow when glucose was present in the のぬな growth medium, but no growth could be observed on any other PTS sugar. Further, non-のぬに PTS sugars, such as raffinose and mellibiose, could stimulate growth of the ptsI mutant のぬぬ (50). It has been suggested that glucose uptake at high growth rates occurs through an のぬね ATP-dependent mechanism and the msm ABC transporter has been proposed as an のぬの alternative glucose transporter (49, 50). However, our results demonstrate that the msm のぬは genes are downregulated, along with PTS activity, when S. mutans UA159 was grown のぬば with excess glucose. Functional analysis of the remaining uncharacterized transport のぬぱ systems upregulated under glucose excess may elucidate whether any of these のぬひ transporters can participate in PTS-independent internalization of glucose or other のねど carbohydrates. のねな

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In an attempt to ascertain the influence of carbohydrate availability on global のねに transcriptional regulation, we searched our microarray data for genetic alterations to the のねぬ CcpA regulon, which is known to include a broad range of transcripts encoding proteins のねね important for the uptake of carbohydrates and pyruvate metabolism. In Gram-positive のねの organisms, CcpA orchestrates gene regulation by binding to a consensus sequence, のねは called a catabolite responsive element (CRE), located near the promoter region of target のねば operons (22, 48). Our results also showed that the co-factor that is known to stimulate のねぱ CcpA binding, HPr(Ser-P), was abundant in cells grown in glucose excess conditions, のねひ whereas we were unable to detect HPr(Ser-P) and HPr(Ser-P)(His~P) in cultures of S. ののど mutans grown with limiting carbohydrate (Figure 4). Using the online bioinformatics tool ののな RegPrecise (http://regprecise.lbl.gov/RegPrecise/), we identified 32 genes within ののに operons downstream of presumptive CREs among the genes that were found to be ののぬ aberrantly regulated in our microarray, which indicated that changes in expression of ののね approximately 18% of the genes affected in this study may be attributed, at least in part, ののの to CcpA (Table 4). Thus, HPr itself, and in conjunction with CcpA, may contribute to the ののは changes in the transcriptome and phenotype of the cells in response to carbohydrate ののば availability. However, it should be emphasized that the lack of major differences in HPr ののぱ levels or phosphorylation status between with wild-type and manL mutant means that ののひ ManL is a major contributor to modifying the transcriptome and phenotypic behavior of S. のはど mutans in response to glucose availability. We were also able to identify several genes のはな from our microarray study bearing predicted CodY binding sites after searching through のはに RegPrecise and previously-collected microarray data, though there were far fewer genes のはぬ than were predicted for the CcpA regulon (Table 4). Interestingly, our study of the のはね affected regulons revealed that the majority of genes of the CcpA regulon were のはの upregulated under glucose limiting conditions, whereas the majority of genes predicted のはは to be under CodY regulation displayed elevated transcription when cells were grown with のはば

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excess glucose. These data indicate that CcpA and CodY may coordinate responses to のはぱ carbohydrate availability and amino acid limitation. のはひ

It would be of interest to perform a comprehensive transcriptional analysis of the のばど manL deletion strain grown under continuous culture conditions with limiting and excess のばな carbohydrate concentrations. Unfortunately, the Pathogen Functional Genomics のばに Resource Center no longer provides microarrays for S. mutans. With the remaining set のばぬ of microarray slides we did have on hand, we used RNA extracted from the manL のばね mutant strain grown under continuous culture with 10 mM and 100 mM glucose to see のばの whether we could find significant changes in the transcriptome in the mutant background のばは in response to carbohydrate availability. We were not satisfied with the quality or with the のばば coverage in these experiments. Consequently, we could not develop a comprehensive のばぱ comparison of the parental and manL mutant strains and have not included the data in のばひ this communication. However, in cases where a sufficient amount of quality data was のぱど obtained for a subset of genes, a small set of genes was found to be differentially のぱな regulated (P < 0.001) in the manL mutant compared to the wild-type strain in response to のぱに carbohydrate availability, and the results were confirmed using qRT-PCR. Since the のぱぬ results are of interest, the genes found to be differentially regulated by the microarray のぱね and confirmed by qRT-PCR have been included in the supplementary data (Table S1), のぱの with the caveat that this does not represent the totality of differences in the transcription のぱは profiles of UA159 and its manL mutant. Recently, our laboratory demonstrated that RNA-のぱば Seq is well-suited for transcription profiling in S. mutans (14), so we plan to more のぱぱ thoroughly probe the extent of the ManL regulon as a function of carbohydrate のぱひ availability using chemostat culture and RNA-Seq. のひど

The levels of EI and HPr changed very little in both wild-type and manL deletion のひな strains in response to the conditions we tested (Figure 3). We noted that the total protein のひに

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level of HPr remained relatively stable between conditions and strains tested, similar to のひぬ what was reported previously (31), and that the levels of ptsH transcript were not のひね significantly altered (Figure S2). Although some studies have demonstrated a four- or のひの five-fold drop in EI levels when cultures were grown in continuous culture with nitrogen のひは limitation (carbohydrate excess) (31, 32), we observed that the amount of EI protein and のひば ptsI mRNA were not substantially altered in response to changes in carbohydrate のひぱ availability (Figure S2). Thus, data from our study do not support the idea that major のひひ changes in the amount of EI or HPr proteins influence the regulation of carbohydrate はどど transport. The differences between our results and those of other may arise from the use はどな of different culture conditions or strains. はどに

In summary, by using steady-state continuous culture, we were able to examine はどぬ the effects of carbohydrate availability on the cariogenic human pathogen S. mutans in はどね the absence of confounding effects from pH, growth rate and other influences that are はどの known to affect gene expression in this organism. Moreover, by using strain UA159, we はどは were able to integrate the physiologic data with complete genome sequence and はどば transcriptomic analysis by microarray. In addition, we further characterized the はどぱ physiology of a strain bearing a deletion in the glucose/mannose EIIAB permease gene, はどひ manL, which has been shown to play a dominant role in CCR of genes in S. mutans and はなど to regulate multiple virulence attributes of the organism (20, 25, 45, 51). The results はなな reveal that there are profound changes in the expression of genes and phenotypic はなに properties of the organism that are known to affect its establishment, persistence and はなぬ virulence in response to the amount of carbohydrate in the environment. The studies はなね also enhance the body of evidence that ManL plays major and direct roles in the control はなの of key virulence attributes in response to carbohydrate source and availability. はなは はなば

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Acknowledgements はなぱ This study was supported by DE12236 from the National Institute of Dental and はなひ

Craniofacial Research. ZM was supported by a University of Florida Alumni Fellowship. はにど We thank Matthew Watts, Kinda Seaton and Jeong Nam Kim for helpful discussions and はにな technical advice while performing microarrays. はにに

はにぬ はにね はにの

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26. Zeng L, Burne RA. 2010. Seryl-phosphorylated HPr regulates CcpA-はひぱ independent carbon catabolite repression in conjunction with PTS permeases in はひひ Streptococcus mutans. Mol. Microbiol. 75:1145-1158. ばどど

27. Ellwood DC, Phipps PJ, Hamilton IR. 1979. Effect of growth rate and glucose ばどな concentration on the activity of the phosphoenolpyruvate phosphotransferase ばどに system in Streptococcus mutans Ingbritt grown in continuous culture. Infect. ばどぬ Immun. 23:224-231. ばどね

28. Hamilton IR, Phipps PJ, Ellwood DC. 1979. Effect of growth rate and glucose ばどの concentration on the biochemical properties of Streptococcus mutans Ingbritt in ばどは continuous culture. Infect. Immun. 26:861-869. ばどば

29. Hamilton IR, Ellwood DC. 1978. Effects of fluoride on carbohydrate metabolism ばどぱ by washed cells of Streptococcus mutans grown at various pH values in a ばどひ chemostat. Infect. Immun. 19:434-442. ばなど

30. Hamilton IR. 1977. Effects of fluoride on enzymatic regulation of bacterial ばなな carbohydrate metabolism. Caries Res. 11(Suppl 1):262-291. ばなに

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31. Rodrigue L, Lacoste L, Trahan L, Vadeboncoeur C. 1988. Effect of nutritional ばなぬ constraints on the biosynthesis of the components of the phosphoenolpyruvate: ばなね sugar phosphotransferase system in a fresh isolate of Streptococcus mutans. ばなの Infect. Immun. 56:518-522. ばなは

32. Hamilton IR, Gauthier L, Desjardins B, Vadeboncoeur C. 1989. ばなば Concentration-dependent repression of the soluble and membrane components ばなぱ of the Streptococcus mutans phosphoenolpyruvate: sugar phosphotransferase ばなひ system by glucose. J. Bacteriol. 171:2942-2948. ばにど

33. Hamilton IR, St Martin EJ. 1982. Evidence for the involvement of proton motive ばにな force in the transport of glucose by a mutant of Streptococcus mutans strain ばにに DR0001 defective in glucose-phosphoenolpyruvate phosphotransferase activity. ばにぬ Infect. Immun. 36:567-575. ばにね

34. Keevil CW, McDermid AS, Marsh PD, Ellwood DC. 1986. Protonmotive force ばにの driven 6-deoxyglucose uptake by the oral pathogen, Streptococcus mutans ばには Ingbritt. Arch. Microbiol. 146:118-124. ばにば

35. Burne RA, Wen ZT, Chen YY, Penders JE. 1999. Regulation of expression of ばにぱ the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and ばにひ carbon catabolite repression. J. Bacteriol. 181:2863-2871. ばぬど

36. Burne RA, Chen, Y. M. 1998. The use of continuous flow bioreactors to explore ばぬな gene expression and physiology of suspended and adherent populations of oral ばぬに streptococci. Methods Cell Sci. 20:181-190. ばぬぬ

37. Belli WA, Marquis RE. 1991. Adaptation of Streptococcus mutans and ばぬね Enterococcus hirae to acid stress in continuous culture. Appl. Environ. Microbiol. ばぬの 57:1134-1138. ばぬは

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38. Kornberg HL, Reeves RE. 1972. Inducible phosphoenolpyruvate-dependent ばぬば hexose phosphotransferase activities in Escherichia coli. Biochem. J. 128:1339-ばぬぱ 1344. ばぬひ

39. LeBlanc DJ, Crow VL, Lee LN, Garon CF. 1979. Influence of the lactose ばねど plasmid on the metabolism of galactose by Streptococcus lactis. J. Bacteriol. ばねな 137:878-884. ばねに

40. Vadeboncoeur C, Thibault L, Neron S, Halvorson H, Hamilton IR. 1987. ばねぬ Effect of growth conditions on levels of components of the ばねね phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus ばねの mutans and Streptococcus sobrinus grown in continuous culture. J. Bacteriol. ばねは 169:5686-5691. ばねば

41. Bender GR, Sutton SV, Marquis RE. 1986. Acid tolerance, proton ばねぱ permeabilities, and membrane ATPases of oral streptococci. Infect. Immun. ばねひ 53:331-338. ばのど

42. Bowden GH, Hamilton IR. 1987. Environmental pH as a factor in the ばのな competition between strains of the oral streptococci Streptococcus mutans, S. ばのに sanguis, and "S. mitior" growing in continuous culture. Can. J. Microbiol. 33:824-ばのぬ 827. ばのね

43. Hamilton IR, Lebtag H. 1979. Lactose metabolism by Streptococcus mutans: ばのの evidence for induction of the tagatose 6-phosphate pathway. J. Bacteriol. ばのは 140:1102-1104. ばのば

44. Hamilton IR, Lo GC. 1978. Co-induction of く-galactosidase and the lactose-P-ばのぱ enolpyruvate phosphotransferase system in Streptococcus salivarius and ばのひ Streptococcus mutans. J. Bacteriol. 136:900-908. ばはど

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45. Zeng L, Das S, Burne RA. 2010. Utilization of lactose and galactose by ばはな Streptococcus mutans: transport, toxicity, and carbon catabolite repression. J. ばはに Bacteriol. 192:2434-2444. ばはぬ

46. An FY, Sulavik MC, Clewell DB. 1999. Identification and characterization of a ばはね determinant (eep) on the Enterococcus faecalis chromosome that is involved in ばはの production of the peptide sex pheromone cAD1. J. Bacteriol. 181:5915-5921. ばはは

47. Thevenot T, Brochu D, Vadeboncoeur C, Hamilton IR. 1995. Regulation of ばはば ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and ばはぱ Streptococcus salivarius. J. Bacteriol. 177:2751-2759. ばはひ

48. Deutscher J. 2008. The mechanisms of carbon catabolite repression in bacteria. ばばど Curr. Opin. Microbiol. 11:87-93. ばばな

49. Buckley ND, Hamilton IR. 1994. Vesicles prepared from Streptococcus mutans ばばに demonstrate the presence of a second glucose transport system. Microbiology ばばぬ 140:2639-2648. ばばね

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

51. Abranches J, Chen YY, Burne RA. 2003. Characterization of Streptococcus ばばぱ mutans strains deficient in EIIAB Man of the sugar phosphotransferase system. ばばひ Appl. Environ. Microbiol. 69:4760-4769. ばぱど

ばぱな ばぱに ばぱぬ

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Table 1. Comparison of pH values achieved by S. mutans UA159 and a strain bearing a ばぱね deletion of manL grown in continuous culture using 10 mM versus 100 mM glucose. ばぱの

Exogenous Glucose Added Endogenous Carbohydrate Stores

pH after 2 minutes pH after 30 minutes pH after 2 minutes pH after 30 minutes

UA159 10 mM glc 5.39 ± 0.31 3.50 ± 0.08 7.13 ± 0.00 6.68 ± 0.06

UA159 100 mM glc 6.23 ± 0.08 3.70 ± 0.01 6.99 ± 0.02 6.39 ± 0.20

∆manL 10 mM glc 6.03 ± 0.21 3.88 ± 0.12 N/D* N/D

∆manL 100 mM glc 6.30 ± 0.20 3.76 ± 0.05 N/D N/D

*Not Determined ばぱは ばぱば

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Table 2. Microarray comparing continuous cultures of S. mutans UA159 grown in 10 mM ばぱぱ versus 100 mM glucose. ばぱひ

Gene Common Name Functional Category Fold-

change* p-value

SMU.1422 putative pyruvate dehydrogenase E1 component beta subunit) Energy Metabolism 133.8 4.74E-04

SMU.1423 putative pyruvate dehydrogenase, TPP-dependent E1 component alpha-subunit Energy Metabolism 124.93 4.50E-05

SMU.1495 galactose-6-phosphate isomerase subunit LacB Energy Metabolism 53.9 2.47E-04

SMU.1539 glycogen branching enzyme Energy Metabolism 30.91 6.00E-07

SMU.113 putative fructose-1-phosphate kinase Energy Metabolism 30.5 2.50E-04

SMU.1538 glucose-1-phosphate adenylyltransferase Energy Metabolism 29.33 5.20E-06

SMU.179 hypothetical protein Unknown 27.2 4.20E-06

SMU.115 putative PTS system, fructose-specific IIA component PTS 25.67 1.93E-04

SMU.2038 putative PTS system, trehalose-specific IIABC component PTS 25.3 1.29E-04

SMU.1537 putative glycogen biosynthesis protein GlgD Energy Metabolism 24.42 6.50E-06

SMU.1425 putative Clp proteinase, ATP-binding subunit ClpB Protein Synthesis and Fate 23.25 1.47E-04

SMU.1340 putative surfactin synthetase Cellular Processes 16.67 2.94E-04

SMU.1536 glycogen synthase Energy Metabolism 16.25 2.29E-05

SMU.148 bifunctional acetaldehyde-CoA/alcohol dehydrogenase Energy Metabolism 15.08 6.50E-06

SMU.180 putative oxidoreductase, fumarate reductase Unknown 14.77 2.12E-05

SMU.252 hypothetical protein Hypothetical 14.41 2.38E-04

SMU.1116c hypothetical protein Hypothetical 13.69 8.89E-05

SMU.1879 putative PTS system, mannose-specific component IID PTS 12.01 8.07E-04

SMU.270 ascorbate-specific PTS system enzyme IIC PTS 11.59 4.55E-05

SMU.1535 glycogen phosphorylase Energy Metabolism 10.34 4.54E-05

SMU.870 putative transcriptional regulator of sugar metabolism Transcription Regulator 9.48 8.40E-06

SMU.1117 NADH oxidase (H2O-forming) Energy Metabolism 8.78 3.03E-04

SMU.290 putative L-ascorbate 6-phosphate lactonase Unknown 8.67 2.52E-04

SMU.402 pyruvate formate-lyase Energy Metabolism 8.56 8.21E-05

SMU.500 putative ribosome-associated protein Protein Synthesis and Fate 8.34 8.72E-05

SMU.881 sucrose phosphorylase, GtfA Transport and Binding 8.1 5.10E-06

SMU.1411 hypothetical protein Unknown 7.61 2.27E-05

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SMU.1878 putative PTS system, mannose-specific component IIC PTS 7.45 6.79E-04

SMU.2127 putative succinate semialdehyde dehydrogenase Central Intermediary Metabolism 7.41 5.06E-05

SMU.1843 sucrose-6-phosphate hydrolase Energy Metabolism 7 3.27E-05

SMU.879 multiple sugar-binding ABC transporter, permease protein MsmF ABC Transporter 6.91 4.55E-04

SMU.871 putative fructose-1-phosphate kinase Energy Metabolism 6.56 8.07E-04

SMU.878 multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE ABC Transporter 6.54 1.52E-05

SMU.880 multiple sugar-binding ABC transporter, permease protein MsmG ABC Transporter 6.49 3.94E-05

SMU.882 multiple sugar-binding ABC transporter, ATP-binding protein, MsmK ABC Transporter 6.46 3.26E-05

SMU.1158c hypothetical protein Unknown 6.44 3.10E-04

SMU.1344c putative malonyl-CoA acyl-carrier-protein transacylase Fatty Acid and Phospholipid Metabolism 6.37 3.65E-04

SMU.1877 putative PTS system, mannose-specific component IIAB PTS 5.93 5.50E-04

SMU.883 dextran glucosidase DexB Energy Metabolism 5.82 6.10E-06

SMU.447 hypothetical protein Unknown 5.79 8.70E-04

SMU.1077 putative phosphoglucomutase Energy Metabolism 5.18 1.23E-05

SMU.1574c hypothetical protein Unknown 4.93 2.47E-04

SMU.887 galactose-1-phosphate uridylyltransferase Energy Metabolism 4.71 1.88E-04

SMU.1088 putative thiamine biosynthesis lipoprotein Biosynthesis of Cofactors, Prosthetic Groups and Carriers

4.38 3.17E-05

SMU.2155 hypothetical protein Unknown 4.35 7.50E-04

SMU.872 putative PTS system, fructose-specific enzyme IIABC component PTS 4.08 3.87E-04

SMU.550 putative cell division protein FtsQ (DivIB) Cellular Processes 3.89 7.10E-04

SMU.1591 catabolite control protein A, CcpA Transcription Regulator 3.65 8.13E-05

SMU.1090 hypothetical protein Unknown 3.6 1.42E-04

SMU.1644c hypothetical protein Unknown 3.48 8.21E-05

SMU.549 undecaprenyldiphospho-muramoylpentapeptide beta-N- acetylglucosaminyltransferase

Cell Envelope 3.47 7.84E-04

SMU.82 molecular chaperone DnaK Cellular Processes 3.2 8.73E-04

SMU.131 putative lipoate-protein ligase Protein Synthesis and Fate 3.17 8.36E-04

SMU.1023 oxaloacetate decarboxylase Amino Acid Biosynthesis 3.06 2.79E-05

SMU.1389 hypothetical protein Energy Metabolism 3.03 4.50E-04

SMU.2035 bacteriocin immunity protein Cellular Processes 2.9 1.54E-04

SMU.271 putative PTS system, enzyme IIB component PTS 2.82 5.86E-05

SMU.129 branched-chain alpha-keto acid dehydrogenase subunit E2 Energy Metabolism 2.77 4.81E-04

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SMU.1089 hypothetical protein Unknown 2.77 4.16E-04

SMU.241c putative ABC transporter, ATP-binding protein, amino acid transport system ABC Transporter 2.76 2.63E-05

SMU.20 putative cell shape-determining protein MreC Cell Envelope 2.57 7.70E-06

SMU.1737 (3R)-hydroxymyristoyl-ACP dehydratase Fatty Acid and Phospholipid Metabolism 2.55 2.96E-04

SMU.274 putative L-xylulose 5-phosphate 3-epimerase Energy Metabolism 2.55 6.42E-04

SMU.2005 adenylate kinase Biosynthesis of Nucleotides and Precursors

2.52 4.56E-05

SMU.963c putative deacetylase Energy Metabolism 2.52 5.74E-04

SMU.2053c hypothetical protein Hypothetical 2.44 4.14E-04

SMU.259 putative oligopeptide ABC transporter, ATP-binding protein OppF ABC Transporter 2.43 4.27E-04

SMU.1462c putative oxidoreductase Unknown 2.38 6.91E-04

SMU.2052c hypothetical protein Hypothetical 2.38 1.95E-05

SMU.1619c putative metalloprotease Unknown 2.24 4.02E-04

SMU.580 exodeoxyribonuclease VII large subunit Biosynthesis of Nucleotides and Precursors

2.24 1.26E-04

SMU.812 hypothetical protein Hypothetical 2.24 4.49E-05

SMU.1428c hypothetical protein Unknown 2.23 5.57E-04

SMU.844 hypothetical protein Unknown 2.23 7.17E-04

SMU.1020 putative citrate lyase CilB, citryl-CoA lyase, beta subunit Energy Metabolism 2.22 1.15E-04

SMU.1679c hypothetical protein Unknown 2.21 1.48E-04

SMU.458 putative ATP-dependent RNA helicase Transcription 2.14 2.35E-05

SMU.1542c putative lipid kinase Unknown 2.07 5.12E-04

SMU.1011 putative CitG protein Energy Metabolism 2.06 2.10E-04

SMU.1207 mobilization/cell filamentation proteins Unknown 2.04 5.04E-04

SMU.1229 purine nucleoside phosphorylase Biosynthesis of Nucleotides and Precursors

2.02 3.68E-04

SMU.517 phosphopantetheine adenylyltransferase Cell Envelope 0.49 4.26E-04

SMU.848 hypothetical protein Unknown 0.49 5.87E-05

SMU.1784c membrane-associated Zn-dependent protease Unknown 0.48 8.82E-04

SMU.712 phosphoenolpyruvate carboxylase Central Intermediary Metabolism 0.47 9.51E-04

SMU.318 putative hippurate hydrolase Central Intermediary Metabolism 0.47 8.98E-04

SMU.234 threonine dehydratase Amino Acid Biosynthesis 0.46 4.47E-04

SMU.1623c hypothetical protein Unknown 0.46 5.44E-04

SMU.509 hypothetical protein Unknown 0.46 3.48E-04

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SMU.841 putative aminotransferase Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.45 2.37E-04

SMU.707c putative endolysin Cell Envelope 0.45 8.01E-04

SMU.1076 hypothetical protein Unknown 0.45 3.15E-04

SMU.1745c putative transcriptional regulator Transcription Regulator 0.44 1.11E-04

SMU.1191 6-phosphofructokinase Energy Metabolism 0.44 6.13E-04

SMU.320 putative 5-formyltetrahydrofolate cyclo-ligase Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.44 4.40E-04

SMU.1325 putative ABC transporter, ATP-binding component ABC Transporter 0.44 3.63E-04

SMU.759 putative protease Unknown 0.43 7.03E-04

SMU.321 hypothetical protein Unknown 0.43 4.92E-05

SMU.364 glutamine synthetase type 1, glutamate--ammonia ligase Amino Acid Biosynthesis 0.42 8.31E-05

SMU.330 glutamyl-tRNA synthetase Protein Synthesis and Fate 0.41 2.37E-04

SMU.572 putative tetrahydrofolate dehydrogenase/cyclohydrolase Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.4 1.00E-07

SMU.2121c hypothetical protein Unknown 0.4 6.18E-04

SMU.1685c hypothetical protein Unknown 0.4 5.04E-04

SMU.1930 putative cytoplasmic membrane protein, LemA-like protein Unknown 0.39 3.16E-04

SMU.648 foldase protein PrsA Cellular Processes 0.39 9.99E-05

SMU.235 hypothetical protein Unknown 0.39 1.85E-04

SMU.429c hypothetical protein Hypothetical 0.39 2.74E-04

SMU.337 hypothetical protein Unknown 0.38 7.49E-04

SMU.647 putative methyltransferase Unknown 0.37 3.86E-05

SMU.1824c transcriptional repressor CodY Transcription Regulator 0.37 5.24E-04

SMU.70 threonine synthase Amino Acid Biosynthesis 0.36 2.56E-04

SMU.1789c hypothetical protein Unknown 0.36 8.32E-05

SMU.2067 putative stress response protein, glycosyltransferase involved in cell wall biogenesis

Unknown 0.36 8.49E-04

SMU.761 putative protease Unknown 0.35 3.38E-04

SMU.1140c hypothetical protein Unknown 0.34 2.72E-05

SMU.2032 30S ribosomal protein S2 Protein Synthesis and Fate 0.34 5.67E-04

SMU.1620 putative phosphate starvation-induced protein PhoH Unknown 0.33 5.00E-07

SMU.22 putative secreted antigen GbpB/SagA, putative peptidoglycan hydrolase Cell Envelope 0.33 2.10E-04

SMU.1823 putative pyrazinamidase/nicotinamidase Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.32 7.48E-04

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SMU.2116 putative osmoprotectant amino acid ABC transporter, ATP-binding protein ABC Transporter 0.32 7.36E-04

SMU.469 Holliday junction-specific endonuclease DNA Metabolism and Repair 0.32 5.87E-04

SMU.568 putative amino acid ABC transporter, ATP-binding protein ABC Transporter 0.32 2.59E-04

SMU.1086 thymidine kinase Biosynthesis of Nucleotides and Precursors

0.31 3.90E-04

SMU.1450 putative amino acid permease Unknown 0.31 4.27E-04

SMU.1786 putative undecaprenyl pyrophosphate synthetase Cell Envelope 0.3 2.79E-04

SMU.676 NADP-dependent glyceraldehyde-3-phosphate dehydrogenase Energy Metabolism 0.3 2.19E-04

SMU.474 S-ribosylhomocysteinase Cellular Processes 0.29 4.80E-04

SMU.1189c hypothetical protein Unknown 0.28 4.26E-05

SMU.1429 putative UDP-N-acetylmuramyl tripeptide synthetase MurC Cell Envelope 0.28 6.99E-04

SMU.369c hypothetical protein Unknown 0.28 5.50E-05

SMU.1476c putative GTP-binding protein Unknown 0.27 2.13E-04

SMU.1326 peptide chain release factor 2 Protein Synthesis and Fate 0.26 3.73E-04

SMU.1437 putative UDP-N-acetylglucosamine 2-epimerase Central Intermediary Metabolism 0.25 3.07E-04

SMU.611 ATP-dependent RNA helicase Transcription 0.24 5.39E-05

SMU.516 hypothetical protein Unknown 0.23 4.47E-05

SMU.530c hypothetical protein Unknown 0.22 2.29E-05

SMU.335 argininosuccinate lyase Amino Acid Biosynthesis 0.22 1.30E-04

SMU.210c hypothetical protein Hypothetical 0.22 6.82E-04

SMU.1627 50S ribosomal protein L11 Protein Synthesis and Fate 0.21 3.00E-06

SMU.283 hypothetical protein Hypothetical 0.21 9.62E-04

SMU.1200 30S ribosomal protein S1 Protein Synthesis and Fate 0.2 3.94E-04

SMU.595 dihydroorotate dehydrogenase 1A Biosynthesis of Nucleotides and Precursors

0.2 9.10E-06

SMU.204c hypothetical protein Hypothetical 0.19 6.65E-04

SMU.2157 inosine 5'-monophosphate dehydrogenase Biosynthesis of Nucleotides and Precursors

0.18 6.29E-04

SMU.951 putative amino acid permease Transport and Binding 0.17 2.42E-04

SMU.396 putative glycerol uptake facilitator protein Transport and Binding 0.17 4.60E-06

SMU.1315c putative ATP-binding protein ABC Transporter 0.17 4.12E-05

SMU.195c hypothetical protein Hypothetical 0.17 7.42E-04

SMU.277 hypothetical protein Hypothetical 0.16 1.63E-05

SMU.1658 putative ammonium transporter, NrgA protein Transport and Binding 0.15 3.14E-04

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SMU.336 ribonuclease P Transcription 0.14 7.47E-04

SMU.1098c putative oxidoreductase Unknown 0.14 2.00E-06

SMU.87 hypothetical protein Unknown 0.14 1.51E-04

SMU.30 putative phosphoribosylformylglycinamidine synthase, (FGAM synthase) Biosynthesis of Nucleotides and Precursors

0.14 4.47E-04

SMU.850 hypothetical protein Unknown 0.13 4.87E-04

SMU.285 hypothetical protein Hypothetical 0.13 4.28E-04

SMU.278 hypothetical protein Hypothetical 0.12 9.40E-06

SMU.1626 50S ribosomal protein L1 Protein Synthesis and Fate 0.12 7.59E-04

SMU.1477 tRNA delta(2)-isopentenylpyrophosphate transferase Protein Synthesis and Fate 0.12 3.66E-04

SMU.1946 hypothetical protein Hypothetical 0.12 3.84E-04

SMU.670 aconitate hydratase Energy Metabolism 0.12 9.51E-04

SMU.71 putative cation efflux pump (multidrug resistance protein) Transport and Binding 0.11 6.97E-04

SMU.281 hypothetical protein Hypothetical 0.11 9.34E-04

SMU.913 glutamate dehydrogenase Amino Acid Biosynthesis 0.11 5.83E-05

SMU.86 hypothetical protein Unknown 0.11 9.10E-06

SMU.196c putative transfer protein Unknown 0.098 7.66E-04

SMU.85 phosphomethylpyrimidine kinase Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.089 5.37E-05

SMU.600c hypothetical protein Unknown 0.077 2.02E-04

SMU.1295 adenosine deaminase Biosynthesis of Nucleotides and Precursors

0.067 3.72E-04

SMU.29 phosphoribosylaminoimidazole-succinocarboxamide synthase Biosynthesis of Nucleotides and Precursors

0.049 6.79E-04

SMU.962 putative dehydrogenase Fatty Acid and Phospholipid Metabolism 0.024 2.59E-04

SMU.961 hypothetical protein Unknown 0.023 4.49E-04

SMU.602 putative sodium-dependent transporter Transport and Binding 0.021 8.57E-04

SMU.1827 putative biotin biosynthesis protein Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.02 4.48E-04

*A fold change above 1.00 indicates an upregulation under glucose limiting conditions. ばひど ばひな

ばひに

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Table 3. RT qPCR confirmation of microarray results for S. mutans UA159 grown in 10 ばひぬ mM versus 100 mM glucose. ばひね

Gene Array Fold

Change

p-value RT Fold Change

p-value

SMU.1424 (SMU.1422) 133.8 4.74E-04 3626.28 1.50E-03

SMU.114 (SMU.113) 30.5 2.50E-04 128.36 3.38E-02

SMU.2038 25.3 1.29E-04 14.31 1.61E-02

SMU.1591 3.65 8.13E-05 4.37 4.85E-03

SMU.1437 0.25 3.07E-04 0.15 4.45E-02

SMU.335 0.22 1.30E-04 0.12 4.55E-02

SMU.29 0.049 6.79E-04 0.03 3.41E-02

ばひの ばひは

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Table 4. Genes identified in a microarray of S. mutans UA159 grown in 10 mM versus ばひば 100 mM glucose that reside within an operon predicted to possess a CRE. ばひぱ

Gene Common Name Functional Category Fold

Change

p-value

SMU.1422 putative pyruvate dehydrogenase E1 component beta subunit) Energy Metabolism 133.8 4.74E-04

SMU.1423 putative pyruvate dehydrogenase, TPP-dependent E1 component alpha-subunit Energy Metabolism 124.93 4.50E-05

SMU.113 putative fructose-1-phosphate kinase Energy Metabolism 30.5 2.50E-04

SMU.179 hypothetical protein Unknown 27.2 4.20E-06

SMU.115 putative PTS system, fructose-specific IIA component PTS 25.67 1.93E-04

SMU.2038 putative PTS system, trehalose-specific IIABC component PTS 25.3 1.29E-04

SMU.180 putative oxidoreductase, fumarate reductase Unknown 14.77 2.12E-05

SMU.1879 putative PTS system, mannose-specific component IID PTS 12.01 8.07E-04

SMU.270 ascorbate-specific PTS system enzyme IIC PTS 11.59 4.55E-05

SMU.870 putative transcriptional regulator of sugar metabolism Transcription Regulator 9.48 8.40E-06

SMU.402 pyruvate formate-lyase Energy Metabolism 8.56 8.21E-05

SMU.500 putative ribosome-associated protein Protein Synthesis and Fate 8.34 8.72E-05

SMU.1878 putative PTS system, mannose-specific component IIC PTS 7.45 6.79E-04

SMU.2127 putative succinate semialdehyde dehydrogenase Central Intermediary Metabolism 7.41 5.06E-05

SMU.1843 sucrose-6-phosphate hydrolase Energy Metabolism 7 3.27E-05

SMU.879 multiple sugar-binding ABC transporter, permease protein MsmF ABC Transporter 6.91 4.55E-04

SMU.871 putative fructose-1-phosphate kinase Energy Metabolism 6.56 8.07E-04

SMU.878 multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE ABC Transporter 6.54 1.52E-05

SMU.880 multiple sugar-binding ABC transporter, permease protein MsmG ABC Transporter 6.49 3.94E-05

SMU.1877 putative PTS system, mannose-specific component IIAB PTS 5.93 5.50E-04

SMU.1077 putative phosphoglucomutase Energy Metabolism 5.18 1.23E-05

SMU.1088 putative thiamine biosynthesis lipoprotein Biosynthesis of Cofactors, Prosthetic Groups and Carriers

4.38 3.17E-05

SMU.872 putative PTS system, fructose-specific enzyme IIABC component PTS 4.08 3.87E-04

SMU.1591 catabolite control protein A, CcpA Transcription Regulator 3.65 8.13E-05

SMU.1389 hypothetical protein Energy Metabolism 3.03 4.50E-04

SMU.271 putative PTS system, enzyme IIB component PTS 2.82 5.86E-05

SMU.274 putative L-xylulose 5-phosphate 3-epimerase Energy Metabolism 2.55 6.42E-04

SMU.1191 6-phosphofructokinase Energy Metabolism 0.44 6.13E-04

SMU.1824c transcriptional repressor CodY Transcription Regulator 0.37 5.24E-04

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SMU.1823 putative pyrazinamidase/nicotinamidase Biosynthesis of Cofactors, Prosthetic Groups and Carriers

0.32 7.48E-04

SMU.611 ATP-dependent RNA helicase Transcription 0.24 5.39E-05

SMU.396 putative glycerol uptake facilitator protein Transport and Binding 0.17 4.60E-06

ばひひ ぱどど

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Figure Legends ぱどな Figure 1. Glycolytic acidification by S. mutans UA159 and a manL mutant strain grown in ぱどに TV base medium supplemented with either 10 mM or 100 mM glucose to steady state in ぱどぬ continuous culture. Experiments were conducted as described in the Methods section, ぱどね and each data set represents an average of three independent results. The pH of the ぱどの cell suspensions was neutralized to pH 7.2, and the change in pH for S. mutans UA159 ぱどは (A) and a manL mutant strain (C) was monitored over time after the addition of glucose ぱどば to a final concentration of 50 mM. An asterisk represents a P-value of less than 0.05 (by ぱどぱ the Student’s t-test). Additionally, to assess metabolism of intracellular stores of ぱどひ carbohydrate, the pH of wild-type cell suspensions was rapidly neutralized, and the ぱなど change in pH was monitored without the addition of exogenous sugar (B). ぱなな ぱなに Figure 2. S. mutans UA159 and a manL mutant strain grown in continuous culture were ぱなぬ assessed for PTS activity as described in the Methods section using (A) glucose (glc), ぱなね fructose (fru) or mannose (man), or (B) maltose (malt), lactose (lac) or cellobiose (cel) as ぱなの the substrate. Each bar represents the average of three independent results. An asterisk ぱなは represents a P-value of less than 0.05, two asterisks represent a P-value of less than ぱなば 0.01, and three asterisks represent a P-value of less than 0.001 (by the Student’s t-test). ぱなぱ ぱなひ Figure 3. Immunoblotting of lysates to determine the levels of ManL, EI and HPr. A) S. ぱにど mutans UA159 grown in continuous culture using TV media supplemented with 10 mM ぱにな or 100 mM glucose were lysed by mechanical disruption and equal amounts of protein ぱにに were separated by SDS-PAGE. Immunoblotting was performed using a ManL, EI, and ぱにぬ HPr antibody at a previously-determined optimal dilution of primary antibody. B) The ぱにね

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manL deletion strain grown in continuous culture using TV media supplemented with 10 ぱにの mM or 100 mM glucose was lysed by mechanical disruption, and equal amounts of ぱには protein were separated by SDS-PAGE. Immunoblotting was performed using EI and HPr ぱにば antibodies. The images are a representative of at least three independent replicates with ぱにぱ each showing similar results. ぱにひ Figure 4. S. mutans UA159 and a manL deletion strain grown in continuous culture using ぱぬど TV medium supplemented with 10 mM or 100 mM glucose were lysed by mechanical ぱぬな disruption and equal amounts of protein were either boiled (indicated by the plus symbol) ぱぬに at 100oC for 5 minutes or immediately subjected to non-denaturing PAGE (indicated by ぱぬぬ the minus symbol) to separate the various species of HPr based on charge. ぱぬね Immunoblotting was performed using an HPr antibody, and the image is a representative ぱぬの of more than three independent replicates with each showing similar results. ぱぬは ぱぬば

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