Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms

Catherine Rathsam, Ruth E. Eaton, Christine L. Simpson, Gina V. Browne, Tracey Berg{dagger}, Derek W. S. Harty and N. A. Jacques

Institute of Dental Research, Westmead Millennium Institute and Westmead Centre for Oral Health, PO Box 533, Wentworthville, NSW 2145, Australia

Correspondence
N. A. Jacques
njacques{at}dental.wsahs.nsw.gov.au


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0·050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.


Abbreviations: 2D, 2-dimensional; 2-DGE, 2-dimensional gel electrophoresis; CcpA, catabolite control protein A; CSP, competence-stimulating peptide; D, dilution rate; IPG, immobilized pH gradient; MALDI, matrix-assisted laser desorption ionization; PMM, peptide mass mapping; TOF, time-of-flight

{dagger}Present addrss: Plant Protection, Elizabeth Macarthur Agricultural Institute, NSW Agriculture, Camden, NSW 2570, Australia.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
It is now well recognized that many species of bacteria preferentially grow as biofilms on surfaces as part of their life cycle (Jefferson, 2004; Stoodley et al., 2002). For the dental caries pathogen, Streptococcus mutans, this is indeed the case as this Streptococcus is not found in high numbers in the oral cavity of edentulous individuals (Lindquist & Emilson, 2004) and is considered an ‘obligate’ biofilm-forming bacterium (Wen & Burne, 2002). In mature dental plaque, oral streptococci possess slow growth rates similar to other bacteria growing on surfaces (Jefferson, 2004), with in vitro mono-culture models of S. mutans showing doubling times in the order of 160 h after 3 days accumulation (Welin et al., 2003). Such a growth rate is 23-fold slower than planktonic cells growing in continuous culture at a dilution rate (D) of 0·1 h–1, a condition used as a control for comparative physiological and/or phenotypic analyses in a number of in vitro studies (Iwami et al., 1992; Jayaraman et al., 1997; Lemos et al., 2001; Len et al., 2003).

Of particular note is the observation that S. mutans maintains a state of competence and transformation efficiency for up to 40 h in mature biofilms (Li et al., 2002), a situation that contrasts markedly with the transient phenomenon observed in batch culture (for reviews see Claverys & Martin, 2003; Cvitkovitch et al., 2003). For example, the genes associated with competence in Streptococcus pneumoniae are transiently expressed over a period of 30–40 min. Recently, the genes associated with competence-stimulating peptide (CSP)-stimulated competence in S. pneumoniae have been shown to exhibit four temporally distinct expression profiles, being classified as ‘early’, ‘late’, ‘delayed’ or ‘repressed’, even though many of the genes so regulated have been eliminated from having a direct role in competence and recombination (Dagkessamanskaia et al., 2004; Peterson et al., 2004). Among the increased pool of mRNA associated with the induction of ‘delayed’ genes in S. pneumoniae are transcripts encoding a number of molecular chaperones and proteinases associated with stress-induced conditions. These proteins have not been eliminated from a role in competence (Dagkessamanskaia et al., 2004; Peterson et al., 2004; Suntharalingam & Cvitkovitch, 2005). As the biofilm state in dental plaque is the primary mode of existence for S. mutans, it is unlikely that it would be preferentially growing under a self-imposed heightened state of stress on a tooth surface in order to maintain a state of competence. Despite this, recent reviews have argued that such a situation is an appropriate response to biofilm formation (Beloin & Ghigo, 2005; Lemos et al., 2005). This conclusion is based on numerous studies, where, for the most part, isogenic mutants have been studied in less than ideal biofilm models without considering possible environmental or pleiotropic effects (Beloin & Ghigo, 2005; Lemos et al., 2005). To further investigate this hypothesis, a comparison has been made of the proteome of S. mutans growing in a mature (48 h) biofilm with that of planktonic cells growing at a steady state in a chemostat. Changes in the level of protein expression have been analysed in relation to alterations in metabolic and anabolic functions relevant to the mature biofilm with particular reference to the extended maintenance of transformation observed in S. mutans biofilms (Li et al., 2001a).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Chemicals and enzymes.
Amino acids, vitamins, adenine, guanine, uracil, mutanolysin, 2-mercaptoethanol, TCA, formic acid, acetone, bacterial protease inhibitor cocktail, chloramphenicol, exonuclease III, melezitose, DTT, thiourea, caprylyl sulfobetaine (N-decyl-N,N-dimethyl-3-ammonia-1-propane-sulfonate), urea, carrier ampholytes pH 3–10, CHAPS, Tris, tributylphosphine, bromophenol blue, Coomassie brilliant blue G, Triton X-100, iodoacetamide, Cellular and Organelle Membrane Solubilizing Reagent, NH4HCO3, mucin and {alpha}-cyano-4-hydroxycinnamic acid were obtained from Sigma–Aldrich. PBS (Dulbecco ‘A’ tablets), pH 7·3, was obtained from Oxoid. Acrylamide solution (40 %), N,N'-methylenebisacrylamide, immobilized pH gradient (IPG) buffers (pH 4·5–5·5, 5·5–6·7, 6–11) and Immobiline DryStrip gels were obtained from Amersham Biosciences. SyproRuby fluorescent dye was sourced from Molecular Probes. Acetonitrile was acquired from Mallinckrodt, modified porcine sequencing grade trypsin from Promega and horse blood from Amyl Media. All other reagents were of analytical reagent grade or the highest purity available.

Bacterial strain, media and growth conditions.
Cells from triplicate continuous cultures of S. mutans LT11 (Tao et al., 1993) grown at 37 °C to a steady state at pH 7·0±0·1 and at a dilution rate (D) of 0·100±0·001 h–1 under anaerobic conditions (95 % N2/5 % CO2) (Jacques et al., 1979) were used as an inoculum for biofilm-grown cells as well as for supplying control planktonic cultures for subsequent comparative proteome analyses. The use of a chemostat greatly reduced the biological variation that is inherent when growing bacteria in batch culture (Len et al., 2004a, b).

To obtain biofilm-grown cells, a temperature-controlled flow reactor with a working volume of 80 ml was constructed in which were suspended two saliva-conditioned glass slides (76·2x25·4 mm; see below; Fig. 1). Medium at pH 7·0 was pumped into the bottom of the reactor and passed out at the top at a standard flow rate of 1·75 mm min–1 over the glass surfaces to mimic the flow rate of saliva over oral tissues (Dawes et al., 1989). A number of these flow reactors maintained at 37 °C were used in any one experiment to obtain sufficient biofilm-grown cells for 2-dimensional electrophoretic (2-DGE) analyses.



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Fig. 1. Schematic diagram of the flow reactor containing two saliva-conditioned glass slides on which biofilms of S. mutans were allowed to form for 48 h prior to harvest.

 
To establish biofilms on saliva-conditioned glass slides, planktonic cells from a matched chemostat were used. Cells were collected directly from the continuous culture vessel and rapidly diluted to an OD600 of 0·500 at 37 °C in fresh culture medium at pH 7·0 prior to inoculating the flow reactor to give a final OD600 of 0·010. After 1 h at 37 °C, medium flow was established in the reactor vessel and the flow maintained for a further 47 h. The source of medium for both planktonic and biofilm cells was a modified nutritional analogue of saliva, DMM (Sissons et al., 1991). The modifications included the use of 25 mM glucose as the source of carbon, alteration in the concentrations of KH2PO4 and K2HPO4 to 15 mM, mucin to 0·0625 % (w/v) and the addition of adenine, guanine and uracil at 20 µg ml–1.

Preparation of saliva-conditioned surfaces.
Mixed human saliva used for conditioning glass slides was obtained from four male non-smoking volunteers who did not have active caries and had healthy periodontal tissues (Macgregor, 1989). Male donors were used to avoid hormonal and other salivary changes during the menstrual cycle in females that might alter the constituents of saliva (Boyer & France, 1976; Choe et al., 1983). Saliva from the four volunteers was collected on ice no earlier than 2 h after the last intake of food and/or beverages and/or oral hygiene measures in a manner approved by Western Sydney Area Health Service, Human Research Ethics Committee, Westmead, New South Wales, Australia. Equal volumes of saliva were pooled to average out differences in salivary components and the mixed saliva was stirred slowly on ice for 10 min in the presence of 2·5 mM DTT prior to centrifugation (50 000 g, 20 min, 4 °C). The supernatant was then filter-sterilized through a 0·2 µm PES filter (Nalge Nunc International) and the glass slides were conditioned by suspending them in the sterilized saliva at 37 °C for 90 min (Palmer & Caldwell, 1995).

Preparation of cellular proteins.
At steady state, the contents of the continuous culture vessel were harvested by centrifugation (10 000 g, 15 min, 4 °C) in the presence of 50 µg chloramphenicol ml–1 and 2·5 µg Sigma proteinase inhibitor cocktail ml–1, and used as a source of planktonic cells. The harvested cells were washed twice in cold PBS (pH 7·8, 4 °C) containing the same inhibitors and the cell pellet was lyophilized. Biofilms of S. mutans growing on saliva-conditioned glass slides were placed on ice and scraped from their substratum using a sterile plastic spatula into cold PBS (pH 7·8, 4 °C) containing the same concentrations of chloramphenicol and proteinase inhibitors used for harvesting planktonic cells. Biofilm cells were then centrifuged, washed and lyophilized in the same manner as planktonic cells. Both planktonic and biofilm cells were processed simultaneously as a set to ensure consistency. A total of three sets of cells derived from three separate chemostats were prepared in this manner.

Aliquots of 20 mg lyophilized planktonic or biofilm cells were partitioned into ‘wall’, ‘cytoplasmic’ and ‘membrane’ fractions using a modification of the method described by Jonquières et al. (1999) to give three protein fractions from equivalent quantities of cells. In essence, each aliquot of cells was resuspended in 1 ml 600 mM melezitose containing 10 mM K2HPO4, 10 mM MgCl2, 1 mM CaCl2, 50 µg Sigma proteinase inhibitor cocktail ml–1 and 2000 U mutanolysin ml–1. The cell suspension was incubated at 37 °C for 60 min and then at 60 °C for 20 min to hydrolyse the S. mutans cell wall and allow the formation of protoplasts. The protoplasts were separated from the released wall-associated proteins by centrifugation (15 000 g, 15 min, 4 °C) and resuspended in 1 ml 600 mM melezitose containing 50 µg Sigma proteinase inhibitor cocktail ml–1. Following recentrifugation (15 000 g, 15 min, 4 °C), the two supernatants containing ‘wall’ proteins were then pooled and dialysed three times against 4 l 18 M{Omega} H2O at 4 °C for 2 h to remove the melezitose. The ‘wall’ proteins were then precipitated with 15 % (w/v) TCA prior to storage for 16 h at –20 °C.

The protoplasts were subsequently resuspended in 700 µl 50 mM Tris/HCl, pH 7·2, containing 10 mM MgCl2, 0·3 % (w/v) DTT and 50 µg Sigma proteinase inhibitor cocktail ml–1 and sonicated (Branson 450; 130 W, 4x15 s, 4 °C with cooling on ice between each burst). The ‘membrane’ fraction was separated from the ‘cytoplasmic’ fraction by centrifugation (21 000 g, 15 min, 4 °C). The ‘membrane’ fraction was resuspended in the same buffer containing Sigma proteinase inhibitor cocktail, recentrifuged (21 000 g, 15 min, 4 °C) and stored at –20 °C. The two supernatants containing ‘cytoplasmic’ proteins were then pooled and precipitated with 15 % (w/v) TCA prior to storage for 16 h at –20 °C.

After thawing, the precipitated ‘wall’ and ‘cytoplasmic’ proteins were recovered by centrifugation (12 000 g, 15 min, 4 °C) and the proteins from each fraction were washed twice in 100 % ice-cold (4 °C) methanol to remove traces of TCA (12 000 g, 15 min, 4 °C) before being allowed to dry in air at room temperature (20–22 °C). All pellets were then stored at –20 °C until required for 2-DGE.

2-DGE.
The precipitated proteins from the ‘wall’, ‘cytoplasmic’ or ‘membrane’ fractions were resuspended in 900 µl of a 2 : 3 mixture of Cellular and Organelle Membrane Solubilizing Reagent and 2-DGE solubilizing solution (modified from Cordwell et al., 2002) and consisting of 5 M urea, 2 M thiourea, 2 % (w/v) CHAPS, 2 % (w/v) caprylyl sulfobetaine, 1·0 % (v/v) Triton X-100, 2 mM tributylphosphine, 0·2 % (v/v) carrier ampholytes (pH 3–10), 40 mM Tris and 0·002 % (w/v) bromophenol blue, sonicated to facilitate resuspension (Branson 450, 130 W, 2x10 s, 4 °C with cooling on ice between each burst) and incubated at room temperature (20–22 °C) for 45 min. Freshly prepared iodoacetamide was then added to each sample to give a final concentration of 15 mM and the incubation continued for a further 45 min at room temperature. The ‘membrane’ fraction was then centrifuged (10 000 g, 10 min, 22 °C) to remove any insoluble material.

Prior to first dimension separation of the proteins, the IPG strips (18 cm, pH 4·5–5·5, 5·5–6·7 and 6–11) were rehydrated overnight at room temperature with 390 µl 2-DGE solubilizing solution. Each of the three fractionated and solubilized protein samples were divided into three equal parts (~300 µl) before addition of one of the IPG buffers of pH 4·5–5·5, 5·5–6·7 or 6–11 to give a final concentration of 1 % (v/v). The protein samples were then loaded onto the corresponding IPG strips using the paper bridge method (Sabounchi-Schutt et al., 2000). Proteins were focused on a Multiphor II (Amersham Biosciences) for a total of 81 kVh (2 h at 100 V, 2 h at 300 V, 2 h at 1000 V, 2 h at 2500 V, 14 h at 3500 V and 5 h at 5000 V), after which the IPG strips were incubated in equilibration buffer [375 mM Tris, pH 8·8, containing 6 M urea, 2 % (w/v) SDS, 20 % (v/v) glycerol, 2·5 % (w/v) acrylamide and 5 mM tributylphosphine] for 10 min (Nouwens et al., 2000). The equilibration buffer was changed once [the second buffer containing 2·5 % (w/v) iodoacetamide] and the IPG strips were incubated for a further 10 min.

The second dimension SDS-PAGE was performed on 12–16 % polyacrylamide gradient gels (~16 h) at 4 °C using an Ettan DALTsix electrophoresis unit (Amersham Biosciences) according to the manufacturer's instructions. Gels were then fixed and stained with SyproRuby for imaging with a Molecular Imager Fx (Bio-Rad) before being ‘double-stained’ with Coomassie brilliant blue G (Cordwell et al., 2002; Len et al., 2003).

Protein isoforms.
The term ‘isoform’ is used here to describe the multiple charged forms of a protein that exist on a given 2-dimensional (2D) gel, where the mean observed Mr for each form calculated from the second (SDS-PAGE) dimension deviated by approximately 10 % or less, and where there was no evidence from peptide-mass mapping (PMM) of some form of truncation or degradation.

Protein identification.
Protein spots excised from the gels were individually destained at room temperature (20–22 °C) in 50 mM ammonium bicarbonate, pH 7·8, containing acetonitrile in the ratio 60 : 40, with some low-peptide-yielding samples being concentrated with C18 Zip-Tips (Millipore). The dried gel pieces were then rehydrated in 2 µl 20 mM ammonium bicarbonate containing 0·004 % trypsin at 4 °C for 60 min. When reswollen, 6–8 µl 18 M{Omega} H2O was added to each gel piece and the samples were incubated for 16 h at 37 °C. An additional 2 µl trypsin solution was then added to each sample and the incubation continued for a further 2 h at 37 °C. Four microlitres of each digested sample was layered in 1 µl aliquots onto a matrix-assisted laser desorption ionization (MALDI) 20x20 sample plate with hydrophobic mask (Micromass). Each aliquot was dried between applications and then 600 nl matrix [3 mg {alpha}-cyano-4-hydroxycinnamic acid ml–1 in 70 % (v/v) acetonitrile] was placed over each spot.

MALDI-time-of-flight (TOF) MS was performed on a Voyager DE-STR (PerSeptive Biosystems) to generate a peptide mass fingerprint of each trypsin-digested protein. All mass spectra were obtained in reflectron-delayed extraction mode. Peak lists were generated using Data Explorer (PerSeptive Biosystems) with trypsin autolysis peptide masses of 842·5 and 2211·1 Da as internal standards. All PMM analyses made use of the program General Protein Mass Analysis for Windows (GPMAW; Lighthouse Data) to search the unpublished single contig of the S. mutans UA159 genome (www.genome.ou.edu/smutans.html) downloaded in May 2002 that was translated in all six reading frames (Ajdic et al., 2002). Parameters for protein identification included a mass tolerance of 50 p.p.m. and a maximum of two missed cleavages per peptide with the methionine sulfoxide and cysteine acrylamide modification options on. Matches were defined on the basis of both >=5 matching peptide masses and >=20 % sequence coverage of predicted Mr and tentatively identified if one but not both of these criteria were met. Protein sequences were then used to query the annotated S. mutans genome at the Oral Pathogens Databases (www.stdgen.lanl.gov/oragen) using the local BLAST search facility to obtain the protein identity. All gene names, corresponding gene numbers, Mr and pI were those on the Oral Pathogen Sequence Database. Protein numbers are shown with the prefix ‘Smut’ and the corresponding gene number from the Oral Pathogen Sequence Database to distinguish them from the gene number at this site which is given with the prefix ‘Smu’. Different protein spots representing high Mr or charged isoforms are given by an alphabetized suffix.

Image analysis.
SyproRuby-stained gels were analysed using the software package z3 (Compugen). Minimum spot area and contrast were set at 50 pixels and 40, respectively, with known scanner artefacts being manually de-selected. Only significant changes in the level of expression of proteins (P<0·050) were considered except where alternative isoforms were identified or where the lack of any change in the level of protein expression was pertinent to the interpretation of the data. The observed Mr and pI co-ordinates on 2D gels were calculated using the z3 software based on the Mr and pI of randomly selected landmark proteins predicted from their gene sequence. Landmark proteins (10–15 per IPG range) were chosen to represent as wide a distribution of co-ordinates on each 2D gel as possible.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
2D displays of fractionated proteins
Fractionation of the cell into the three compartments, ‘cytoplasm’, ‘membrane’ and ‘wall’, coupled with the use of narrow pH range IPG strips (Len et al., 2003), significantly enhanced the number and clarity of protein spots that could be discerned on 2D gels following staining with SyproRuby. However, little partitioning of cytoplasmic proteins into the ‘cytoplasmic’ fraction was apparent with most of the cytoplasmic proteins remaining in the ‘membrane’ fraction (Table 1).


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Table 1. Number of protein spots found on 2D gels

The data show the number of protein spots detected by z3 analysis using the set filter parameters. These parameters would have resulted in the exclusion of very low abundance protein spots that were not stained by Coomassie blue G nor would be detectable by MALDI-TOF. The data represent the total number of protein spots from master gels constructed from overlays of 2D displays of planktonic and biofilm-grown cells and as such include protein spots unique to either growth condition.

 
Of the 347 Coomassie-stained protein spots from the cellular fractions of S. mutans chemostat- and biofilm-grown cells that were analysed, 242 (representing 115 different proteins) met our set criteria for identification following PMM (>20 % coverage and >5 matching peptides). Forty-eight were significantly altered in their level of expression (P<0·050) or were unique to planktonic or biofilm-grown cells (Tables 2 and 3). In all but one case it was noted that where the changes in the mean levels of expression were greater than twofold, these values were not significantly different. As previously reported, the main source of this error was the reproducibility of the 2D displays themselves (Alban et al., 2003; Len et al., 2004a, b).


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Table 2. Non-competence related proteins differentially expressed in S. mutans biofilms

 

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Table 3. S. mutans proteins homologous to CSP-stimulated proteins from S. pneumoniae

Proteins encoded by genes homologous to those in S. pneumoniae which are known to be, or have not been eliminated from, those required for competence and which are altered in their level of expression during CSP-stimulation of S. pneumoniae batch cultures (Peterson et al., 2004).

 
Alterations in the fermentation phenotype of the S. mutans biofilm
It is logical to hypothesize that any slowing of the growth rate in mature biofilms might be reflected in a down-turn in the level of metabolic enzymes, even though care should be taken in interpreting such a reduction as implying a corresponding down-turn in the flux of metabolites (Len et al., 2004b). Notwithstanding, neither this nor previous 2-DGE analyses (Len et al., 2003, 2004b) have identified the integral cytoplasmic membrane glucose transporters EIIman and EIIglc, or the glucose permease of S. mutans required for uptake of glucose (Cvitkovitch et al., 1995; Vadeboncoeur et al., 1991) due to their hydrophobic structure. As a consequence, any change that the level of expression of these transporters may have on glucose uptake and subsequent metabolic rate cannot be surmised. However, a 3·7-fold down-regulation of glucokinase (Glk) required for the ATP-dependent phosphorylation of free intracellular glucose was noted in biofilm cells (Table 2, Fig. 2). Furthermore, unlike previous analyses where only truncated forms of the protein EIIABman (ManL) were observed (Len et al., 2003, 2004b), five isoforms of the protein were identified (Table 2, Fig. 2). Of these, only Smut1707b and Smut1707e were down-regulated to a significant degree (Table 2). However, even though the mean level of ManL was down-regulated overall by a factor of 2·3-fold in biofilm-grown cells, this change was not statistically significant (P<0·206). As with other protein spots discussed below that were identified in more than one isoform, the true nature of these isoforms has not been determined. It is therefore not known whether the five isoforms of ManL were due to the loss of C- and/or N-terminal amino acids (as is likely with Smut1707a and b proteins spots; Table 2), alternative forms of phosphorylation (consistent with the regulatory role of this protein) (Vadeboncoeur & Pelletier, 1997; Abranches et al., 2003) or some form of artefact (Len et al., 2003).



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Fig. 2. General trends in proteins expressed by S. mutans involved in carbon metabolism. The columns represent an estimate only of the total relative mean level of each protein based on planktonic cells grown at pH 7·0 (100 %) after taking into account the various forms of a given protein observed on 2D gels. The data do not take into account the possibility of post-translational modifications or other processing events that may account for alternative isoforms detected on 2D gels. Columns in ovals represent planktonic cells grown at pH 7·0 (black), planktonic cells grown at pH 5·0 (cross-hatched; Len et al., 2004b) or biofilm-grown cells (dots).

 
As well as Glk, another kinase, galactokinase (GalK), was identified on 2D gels, but only in planktonic cells (Table 2). GalK forms part of the Leloir pathway of galactose metabolism (Ajdic et al., 1996) and is induced in the presence of galactose, a constituent of the mucin present in the DMM medium (Herp et al., 1979; Zalewska et al., 2000). The absence of GalK in biofilm cells may be due to carbon catabolite repression of galK by glucose. A lack of planktonic growth in the biofilm reactors implied that the rate of dilution of the medium in these reactors was greater than the maximum specific growth rate, µmax. This observation suggested that glucose might also be in excess in the biofilms themselves, leading to carbon catabolite repression of galK.

In Gram-positive bacteria, carbon catabolite repression is considered to be regulated in part by means of the catabolite control protein A (CcpA) where loss of CcpA results in relief of catabolite repression (Saier et al., 1996). In marked contrast, however, inactivation of the homologous gene, regM, in S. mutans results in increased glucose repression of responsive genes (Simpson & Russell, 1998) and has been reported to reduce the ability of S. mutans to form a biofilm (Wen & Burne, 2002). The cumulative 6·8-fold down-regulation of the four forms of RegM in biofilms of S. mutans (Table 2; P<0·010) was in keeping with such a role for RegM in carbon catabolite repression. The cumulative down-regulation of RegM in S. mutans biofilms should be contrasted with Bacillus subtilis where carbon catabolite repression by glucose, operating through elevated levels of CcpA, inhibits biofilm formation (Stanley et al., 2003). These contrasting results may be explained by the fact that the biofilm state of dental plaque is the preferred state of growth of S. mutans while in B. subtilis, starvation appears to promote the planktonic to biofilm transition (Stanley et al., 2003), emphasizing a need for differences in the mechanism of carbon catabolite repression between these two Gram-positive bacteria.

A general reduction in the levels of expression of protein spots associated with the remaining ten enzymes of glycolysis were noted in biofilm cells, though none were down-regulated more than twofold, nor any in a statistically significant manner (data not shown). The pyruvate formed by glycolysis, however, is converted in S. mutans to a variety of end products by the co-ordinated use of the pyruvate dehydrogenase complex (AcoA and AcoB, EC 1.2.4.1; AcoC, EC 2.3.1.12; AcoL, EC 1.8.1.4), L-lactate dehydrogenase (Ldh, EC 1.1.1.27), pyruvate formate lyase (Pfl, EC 2.3.1.54) and/or acetolactate synthase (AlsS, EC 2.2.1.6) (Len et al., 2004b; Fig. 2). Of the 11 enzymic steps involved in these pathways, two forms of phosphotransacetylase (Pta) were down-regulated 2·3-fold in biofilm-grown cells (Table 3, Fig. 2), while acetoin dehydrogenase (ButA) was up-regulated 3·4-fold (Table 2, Fig. 2). Up-regulation of ButA contrasts with that observed in planktonic cells grown at pH 5·0 where the level of ButA was down-regulated 32-fold by acidic conditions (Len et al., 2004b). The data suggest that biofilm cells may be producing diacetyl rather than acetate. Unfortunately, the nature of the biofilm fermenters used to grow the biofilm cells has so far precluded any analysis of secreted fermentation by-products, due to the extent of dilution from inflowing medium.

ATP-binding proteins
Eight ATP-binding proteins of ABC transporters were identified on 2D gels. Of the four associated with sugar uptake, the ATP-binding protein of an orthologue of the MsmK multiple-sugar-binding transport system of S. mutans associated with the maltose uptake system (MsmK-like protein) was down-regulated 4·3-fold in biofilm-grown cells. Similarly, the ATP-binding proteins of the branched chained amino acid transporter, LivF (Smut1517), an orthologue of the ATP-binding protein of the glutamine transporter GlnQ of Streptococcus agalactiae (Smut0418), and two charged isoforms and a degraded form (Mr 21 270±650, pI 6·2±0·1) of the glycine betaine/choline homologue of S. agalactiae, AtmD (Smut1003), were down-regulated or absent from biofilm-grown cells (Table 2). The other two ATP-binding proteins included a homologue of YurY (Smut1093), a protein of unknown function that was the only ATP-binding protein identified as being up-regulated in biofilm-grown cells (Table 2). The other protein was a homologue of SloA (Smut0164), a protein associated with Mn and Fe uptake in S. mutans encoded by the sloABCR operon. SloA forms part of an ABC transport complex that belongs to the lipoprotein receptor antigen I (LraI) family, as the lipoprotein ion-binding component of this transport system, SloC, is antigenic and a known virulence trait in infective endocarditis (Kitten et al., 2000; Paik et al., 2003). In biofilm-grown cells, SloA was down-regulated 2·3±0·6-fold (Table 2). As to whether the Mn-dependent regulator, SloR, was also down-regulated could not be determined as the data were not statistically significant (Table 2). The SloR protein represses the sloABCR operon in response to a build up in the concentration of cellular Mn (Kitten et al., 2000; Paik et al., 2003) which is used to detoxify a variety of reactive oxygen species in S. mutans (for recent review see Jakubovics & Jenkinson, 2001). Failure to protect against oxygen species can, for example, lead to oxidized abasic lesions in DNA. Such sites are most efficiently repaired by the exonuclease III, ExoA, which has been purified from S. mutans and found to be up-regulated in cells grown at low pH (Greenberg et al., 2004; Hahn et al., 1999). It is reasonable to hypothesize that protection of S. mutans from oxygen species by a build up of cellular Mn should warrant a reduction in ExoA expression, as was the case in biofilm-grown cells where ExoA was down-regulated 4·3-fold (Table 2).

Replication and the cell envelope of S. mutans growing in a biofilm
A general down-turn in metabolic function and cell doubling in biofilm-grown cells should be reflected in a reduced need for DNA synthesis for chromosomal replication. In biofilm-grown cells, a 4·5-fold reduction in the level of transketolase (Tkt) was observed (Table 2). Tkt redirects glycolytic intermediates into the pentose phosphate pathway and ultimately to nucleotide synthesis. This reduction should be contrasted with that occurring in planktonic cells grown at low pH where an increase in Tkt appears to be required to channel carbon into nucleotide synthesis to repair replicative and transcriptional damage due to acidification of the cytoplasm (Len et al., 2004b).

An early step in chromosomal replication requires the delivery of a hexameric annular helicase onto ssDNA to further unwind the dsDNA (Patel & Picha, 2000). In B. subtilis, the recruitment of the monomers of the helicase (DnaC) in the correct topology around the ssDNA is mediated by the helicase loader proteins, DnaI and DnaB (Velten et al., 2003). The DNA helicase loader, DnaI, was identified on 2D gels of S. mutans, but only in planktonic cells (Table 2), suggesting that repression of DnaI occurs in biofilm-grown cells in response to a reduced requirement for chromosomal replication.

Any decrease in the rate of cell doubling should also be reflected in processes associated with cell envelope biosynthesis. In this regard, four of the six enzymes involved in polymerizing acetyl-CoA into fatty acids prior to the synthesis of membrane lipids, FabD, FabF, FabG and FabK, were identified. Multiple forms of two of the enzymes, failure to detect some of these enzymes in biofilm-grown cells and lack of reproducible detection on 2-DGE made any prediction of an overall change in fatty acid synthetic enzymes problematic, even though the data suggested a down-regulation of the enzymes in biofilm-grown cells (Table 2). Indirect support for this view comes from the arrangement of the genes encoding FabD, FabG and FabK that appear to form an operon in S. mutans (www.stdgen.lanl.gov/oragen). The cumulative 2·5-fold decrease in expression of FabG might therefore reflect a down-regulation in FabD and FabK. Also consistent with this hypothesis was the finding that an isoform of NAD-dependent glycerol-3-phosphate dehydrogenase (GpdA), which forms the last step in glycerolipid degradation (Len et al., 2004b), was reduced 4·3-fold in biofilm-grown cells (Table 2, Fig. 2).

A cumulative 6·3-fold reduction in the level of the two charged isoforms of phosphoglucomutase (PgmA) in biofilm-grown cells was also noted (Table 2). PgmA converts glucose 6-phosphate to glucose 1-phosphate which is a substrate for rhamnose biosynthesis, a sugar component of the cell wall polysaccharide of S. mutans (Schleifer & Kilpper-Bälz, 1987). The first two enzymes in this pathway, two isoforms of glucose-1-phosphate thymidylyltransferase (RmlA) and one of dTDP-glucose-4,6-dehydratase (RmlB) were also down-regulated in biofilm-grown cells (Table 2). PgmA can also convert D-glucosamine 6-phosphate to D-glucosamine 1-phosphate, leading to the formation of UDP-N-acetyl-D-glucosamine, a precursor of peptidoglycan and glycolipid biosynthesis. PgmA is the second enzyme in this pathway which involves the initial conversion of D-fructose 6-phosphate to D-glucosamine 6-phosphate by L-glutamine-D-fructose-6-phosphate aminotransferase (GlmS, EC 2.6.1.16). Although the mean values of GlmS and two other enzymes in the pathway, phosphoglucosamine mutase (phosphoacetylglucosamine mutase; GlmM, EC 5.4.2.3) and UDP-N-acetylglucosamine pyrophosphorylase (GlmU, EC 2.7.7.23/2.3.1.157) were down-regulated in biofilm-grown cells by factors of 2·7-, 5·3- and 6·3-fold, respectively, none of these reductions was statistically significant (data not shown).

Also identified on 2D gels were enzymes associated with the diaminopimelate pathway involved in the formation of cell wall cross-linking peptides (Berges et al., 1986). Of the two enzymes identified, dihydrodipicolinate reductase (DapB, EC 1.3.1.26) and tetrahydrodipicolinate succinylase (DapD), DapD was the only one that was down-regulated in a statistically significant manner (Table 2).

Conserved hypothetical proteins
Four conserved hypothetical proteins were altered in their level of expression in biofilm-grown cells. Two of these, Smut0697 and Smut0873, were uniquely expressed in biofilms, while the two charged isoforms of Smut1337 were absent from biofilm-grown cells and the three charged isoforms of Smut1602 were down-regulated 2·8-, 3·4- and 5·9-fold (Table 2).

Expression of competence-related proteins in S. mutans biofilms
Twenty-nine different proteins were identified in S. mutans that were encoded by genes homologous to the recently defined CSP-responsive ‘early’, ‘late’, ‘delayed’ and ‘repressed’ genes of S. pneumoniae. However, 18 of these have been shown to have no involvement with competence per se (Dagkessamanskaia et al., 2004; Peterson et al., 2004). In streptococci, including S. mutans, genetic competence is mediated by quorum sensing in which cell–cell signalling is regulated by CSP (Claverys & Martin, 2003; Cvitkovitch et al., 2003; Kleerebezem et al., 1997; Li et al., 2001a, 2002). None of the six S. mutans quorum sensing proteins, ComA, ComB, ComC, ComD, ComE or ComX, was identified on 2D displays. Their small size, low abundance or integral association with the cytoplasmic membrane would most likely account for this (Len et al., 2003). However, six ‘late’ competence proteins were identified that were predicted to be under the control of the alternative sigma factor ComX ({sigma}x), since they contained putative competence-induced (cin)-boxes in their promoter regions (Peterson et al., 2004). They were the ribosomal interface protein YifA, the metabolic enzyme Pta, involved in acetate formation in the pyruvate formate lyase pathway (see above), the recombinase A protein RecA, an ssDNA-binding protein, SsbA, a DNA-processing protein, Smf, and a conserved hypothetical protein, Smut0697 (see above), which in S. pneumoniae has been shown not to be directly involved in competence (Peterson et al., 2004) (Tables 2 and 3). In S. mutans, the cin-box consensus sequence, TACGAATA, is to be found 20 bp upstream of the recA operon (Martin et al., 1995; Mortier-Barriere et al., 1998) and 21 bp upstream of the Smu0697 gene. recA itself possesses an incomplete cin-box, TA-GAATA, 12 bp upstream of its own ORF in the cinA gene that completes the operon. The yifA gene lies downstream of the comF and comFC genes in S. mutans in an equivalent manner to the organization of the three homologous genes (SP2206, SP2208 and SP2207, respectively) in S. pneumoniae TIGR4 and may form an operon with these genes. A cin-box is found 10 bp upstream of the putative helicase-encoding gene, comF, of S. mutans. On the other hand, the pta gene of S. mutans has a cin-box-like sequence, TACaAAaA, 22 bp upstream, but lying within the pseudouridine synthase gene, rluE. The ssbA gene of S. mutans also has a modified cin-box sequence, TgCGAATA, 27 bp upstream and similar to the modified cin-box sequence, TtCGAATA, 23 bp upstream of the smf gene. This latter cin-box sequence is identical to a putative cin-box sequence in the promoter of a comG1-like gene which has been identified as possibly encoding a ‘late’ competence protein in S. mutans (Cvitkovitch, 2001).

Both YfiA and Pta were down-regulated in biofilm cells by a factor of 2·2- and 2·3-fold, respectively (Table 3), contrary to the expectation that they would be up-regulated as a response to the maintenance of competence (Peterson et al., 2004). However, this was not the case for the other four proteins. Seven of the eight high-Mr charged isoforms of RecA were uniquely expressed in biofilm cells along with the ssDNA-binding protein SsbA and the conserved hypothetical protein Smut0697 (Tables 2 and 3). Although the mean level of the DNA processing protein Smf was upregulated 4·3-fold in biofilm-grown cells, the data were not statistically significant (Table 3).

In S. pneumoniae, RecA, SsbA and the Smf equivalent, DprA, have essential roles in competence. Smf/DprA is required to protect incoming ssDNA from hydrolysis by DNase (Berge et al., 2003; Campbell et al., 1998), while RecA not only protects incoming ssDNA, but also promotes recombination (Berge et al., 2003). The recA operon in S. pneumoniae and S. mutans includes a cinA homologue that encodes a colligrin that is believed to target RecA to the membrane during competence (Masure et al., 1998). This may result in the topological protection of ssDNA as it enters the cell, thus increasing transformation efficiency (Masure et al., 1998). The up-regulation of RecA (and possibly that also of Smf) could therefore constitute a switch controlling the choice between recombination and degradation at the early stage of transformation in streptococci. (Berge et al., 2003). The other role for RecA during competence is recombination (Courcelle & Hanawalt, 2003; Courcelle et al., 2001). Under stress conditions, when damage to chromosomal DNA stalls replication, RecA forms part of the SOS response (Volkert & Landini, 2001; Bjedov et al., 2003). Under these conditions, RecA can also initiate DNA repair by recombination, thus providing an explanation as to why it is up-regulated in S. mutans growing at low pH (Len et al., 2004a). In biofilm-grown cells, however, RecA was observed to be up-regulated to a far higher degree than in planktonic cultures at low pH (Len et al., 2004a; Fig. 3).



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Fig. 3. General trends in proteins expressed by S. mutans involved in replication, transcription and translation. The columns represent an estimate only of the total relative mean level of each protein based on planktonic cells grown at pH 7·0 set at 100 % after taking into account the various forms of a given protein observed on 2D gels. The data do not take into account the possibility of post-translational modifications or other processing events that may account for alternative isoforms detected on 2D gels. Columns in ovals represent planktonic cells grown at pH 7·0 (black), planktonic cells grown at pH 5·0 (cross-hatched; Len et al., 2004a), biofilm-grown cells (dots) or uniquely expressed in biofilm cells (horizontal dashes). A protein spot corresponding to Smf was not detected in planktonic cells grown at pH 5·0 (Len et al., 2004a).

 
One of the two ssDNA-binding proteins synthesized by S. mutans, SsbA, was also up-regulated in biofilm-grown cells (Table 3). Homologues of the ssbA gene in both S. pneumoniae (SP1908) and B. subtilis (BS0650) are considered to be ‘late’ competence genes, the products of which have a possible role of stabilizing ssDNA during the recombination step of transformation (Campbell et al., 1998; Dubnau, 1991; Lindner et al., 2004; Peterson et al., 2004; Steffen et al., 2002). The other ssDNA-binding protein expressed by S. mutans, Ssb, is up-regulated by pH stress (Len et al., 2004a) but down-regulated in biofilm-grown cells (Table 2, Fig. 3). This observation is similar to that recently reported for B. subtilis where SsbA is considered to be involved in binding ssDNA during transformation, while Ssb is associated with the SOS response (Lindner et al., 2004). In fact, in S. pneumoniae, Ssb appears to impede recombination by limiting the efficiency of the DNA strand exchange reaction by displacing RecA from ssDNA when Ssb levels are high (Steffen et al., 2002). Such observations raise the possibility that association with either of the two ssDNA-binding proteins could modify the RecA response, with Ssb driving RecA repair during stress and SsbA driving RecA recombination during transformation.

Under stress conditions, such as those imposed by planktonic growth at low pH, not only are the levels of RecA and Ssb elevated, but also those of other stress-related proteins, the genes for which are classified as ‘delayed’ competence genes in S. pneumoniae (Dagkessamanskaia et al., 2004; Peterson et al., 2004). These proteins include the molecular chaperones DnaK, GroEL and GroES. Contrary to the observations of gene expression in planktonic batch cultures during CSP-induced competence in S. pneumoniae, none of these three proteins was elevated in S. mutans biofilms (Table 3, Fig. 4). These data for biofilm-grown cells also contrast with the proteome of S. mutans grown at low pH where the molecular chaperones are up-regulated (Len et al., 2004a). This observation is borne out by various molecular biological studies which show that mutants lacking the proteins or the ability to increase the level of molecular chaperones at low pH are more susceptible to acid killing (for recent review, see Lemos et al., 2005).



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Fig. 4. General trends in molecular chaperones and proteinases expressed by S. mutans. The columns represent an estimate only of the total relative mean level of each protein based on planktonic cells grown at pH 7·0 (100 %) after taking into account the various forms of a given protein observed on 2D gels. The data do not take into account the possibility of post-translational modifications or other processing events that may account for alternative isoforms detected on 2D gels. Columns in ovals represent planktonic cells grown at pH 7·0 (black), planktonic cells grown at pH 5·0 (cross-hatched; Len et al., 2004a), biofilm-grown cells (dots) or uniquely expressed planktonic cells at pH 5·0 (horizontal dashes; Len et al., 2004a). Protein spots corresponding to PepB, PepD and RopA were not identified in the current study.

 
Another molecular chaperone, RopA, the protease PepD and the transcriptional factors RpoA and GreA were identified as being elevated during acid-tolerant growth in S. mutans (Len et al., 2004a). However, only the two transcriptional factors were identified in the current study. Two high-Mr forms of RpoA were identified; Smut1817a, being down-regulated 3·0-fold, and Smut1817b being up-regulated 3·3-fold in biofilm-grown cells (Table 2). The combined total of the two forms, however, was down-regulated 2·5-fold (P<0·002) in biofilm-grown cells, since the higher Mr form, Smut1817a, was present at a 34±9·0-fold greater level than Smut1817b on 2D gels from planktonic cells (Fig. 3). Although GreA was down-regulated 1·9-fold in biofilm cells, the data were not significant (P<0·054).

Concluding remarks
While it cannot be precluded that the slower rate of growth of S. mutans in a mature biofilm might influence the level of expression of proteins compared with those expressed in planktonic cells grown at a D of 0·1 h–1, the comparative proteome analysis of these two states, surrounded as they were by a neutral pH environment, was consistent with the hypothesis that the slower growing mature biofilm phenotype was a regulated state and not subject to a stress-induced response arising from nutrient limitation (Lemos et al., 2005). The lack of enhanced levels of molecular chaperones and other stress-related proteins supported this contention. Whether the elevated levels of these ‘delayed’ competence genes observed in batch cultures of other streptococci are essential for competence remains to be determined.

The observed increase in the level of the competence protein, RecA, however, would allow S. mutans to more rapidly initiate an SOS response in a biofilm following a rapid drop in pH as occurs naturally in the oral cavity during the intake of dietary carbohydrates and which is an inherent consequence of oral biofilms studied in a closed environment such as the microtitre biofilm models used in the majority of studies reviewed by Lemos et al. (2005). The maintenance of high levels of RecA may be one factor that explains why S. mutans biofilms are more resistant to a sudden exposure to low pH, a phenomenon known as the acid-tolerant response. This may also have a bearing on the apparent relation of the acid-tolerant response to actively transcribed competence genes (Cvitkovitch et al., 2003; Li et al., 2001b). Whatever the situation, the data obtained in this study provide the first direct evidence for the presence of proteins needed to explain the extended time-frame for transformation in S. mutans growing in a biofilm (Li et al., 2001a). As to whether the maintenance of competence in biofilms allows DNA to be assimilated from the external environment primarily as a nutrient source or to increase genetic diversity by recombination remains to be determined (Jefferson, 2004; Claverys et al., 2000). The fact that only 0·03 % of S. mutans are transformed in biofilms (Aspiras et al., 2004) may be evidence in favour of the former proposal and a reassessment of the true function of competence is required in this and other bacteria, particularly when growing in their natural sessile state as biofilms.


   ACKNOWLEDGEMENTS
 
This research was supported by grant no. R01 DE 013234 from the Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), USA. We wish to thank Professor Phil Robertson and Valentina Valova of the Children's Medical Research Institute, Westmead, for access and use of their mass spectrometry facilities as well as the continued assistance of the Australian Proteome Analysis Facility (APAF), established under the Australian Government Major National Research Facility programme. Special thanks are also given to Helen Dalton, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, for establishing the parameters for the colonization of surfaces by S. mutans.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 14 December 2004; revised 3 March 2005; accepted 4 March 2005.



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