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
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ABSTRACT |
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Present addrss: Plant Protection, Elizabeth Macarthur Agricultural Institute, NSW Agriculture, Camden, NSW 2570, Australia.
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INTRODUCTION |
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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 3040 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
).
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METHODS |
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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 h1 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 min1 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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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 ml1 and 2·5 µg Sigma proteinase inhibitor cocktail ml1, 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 ml1 and 2000 U mutanolysin ml1. 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 ml1. 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
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 ml1 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 (2022 °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 310), 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 (2022 °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 4·55·5, 5·56·7 and 611) 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 4·55·5, 5·56·7 or 611 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 1216 % 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 (2022 °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, 68 µl 18 M 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
-cyano-4-hydroxycinnamic acid ml1 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 (1015 per IPG range) were chosen to represent as wide a distribution of co-ordinates on each 2D gel as possible.
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RESULTS AND DISCUSSION |
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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 cellcell 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 (
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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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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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 h1, 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 December 2004;
revised 3 March 2005;
accepted 4 March 2005.
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