Two-Dimensional Fluorescence Difference Gel Electrophoretic Analysis of Streptococcus mutans Biofilms Catherine Rathsam,† Ruth E. Eaton,† Christine L. Simpson,† Gina V. Browne,† Valentina A. Valova,‡ Derek W. S. Harty,† and Nicholas A. Jacques*,† Institute of Dental Research, Westmead Millennium Institute and Westmead Centre for Oral Health, Westmead, New South Wales, Australia and Children’s Medical Research Institute, Westmead, New South Wales, Australia Received July 29, 2005
Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 18231837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated. Keywords: proteomics • two-dimensional fluorescence difference gel electrophoresis (DIGE) • Streptococcus mutans • biofilm • stress-responsive proteins • competence • quorum sensing • metabolism
Introduction Biofilms are important in human disease as 65% of microbial infections are caused by bacteria growing on surfaces rather than in the free living planktonic state. In the biofilm mode of growth, microorganisms exhibit increased resistance to antimicrobial compounds, environmental stresses, and host immune defense mechanisms.1 In the oral cavity, microbial biofilms account for the two most prevalent pandemic diseases of mankind, dental caries and periodontal diseases, both of which have a high rate of morbidity and represent a significant economic burden to society. Oral biofilms on teeth are usually in equilibrium with the host and are therefore beneficial since they prevent colonization by exogenous and potentially pathogenic species. However, environmental perturbation of the biofilm can lead to an overgrowth of virulent species that form a part of the natural polymicrobial microflora of dental plaque. The current working hypothesis for the initiation of dental caries, the ecological plaque hypothesis, states that disease follows unfavorable disruption of the dynamic balance between the host and the microbial community at local sites.2 In * To whom correspondence should be addressed. Institute of Dental Research, Westmead Centre for Oral Health, PO Box 533, Wentworthville, NSW 2145, Australia. Tel: +612-9845-8763. Fax: +612-9845-7599. E-mail:
[email protected]. † Institute of Dental Research, Westmead Centre for Oral Health, PO Box 533, Wentworthville, NSW 2145, Australia. ‡ Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145 Australia. 10.1021/pr0502471 CCC: $30.25
2005 American Chemical Society
particular, extended periods of dental plaque acidification results in the emergence of acidogenic and aciduric bacteria that can demineralize tooth enamel. Two species of mutans streptococci, Streptococcus mutans and Streptococcus sobrinus, are considered the primary etiological agents of human dental caries due to their elevated numbers in cariogenic plaque and their cariogenic status in experimental animals.3 S. mutans, however, has been the primary focus of study due to its strong association with dental caries in developed nations and its natural ability to be transformed. Inherent in the ecological plaque hypothesis is the concept that disease can be controlled not only by targeting pathogens but also by interfering with the factors responsible for driving the deleterious shifts. Such theories require an holistic, or Systems Microbiological approach, to study disease as they are not tractable by traditional approaches alone. A 2004 report by the American Academy of Microbiology (http://www.asm.org/academy) emphasized the technical bottlenecks associated with the systems approach, including the need to obtain meaningful quantitative proteome data. Acquisition of statistically significant quantitative data from two-dimensional (2D) displays, however, is inherently difficult using traditional 2D electrophoretic approaches, even when using techniques to minimize biological error, such as growing planktonic cultures of S. mutans at steady state and using these bacteria as controls for comparative proteome analysis of biofilm grown cells. This is particularly the case for enzymes associated with general Journal of Proteome Research 2005, 4, 2161-2173
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Published on Web 11/02/2005
research articles metabolic functions such as glycolysis and alternative acid production.4 Here we report the use of 2D fluorescence difference gel electrophoresis (DIGE) to overcome difficulties in determining the extent of biological and experimental error associated with comparative analyses using a conventional 2D approach, particularly those errors associated with the reproducibility of the 2D displays themselves.5 DIGE was first described by Unlu’s laboratory6 and subsequently developed by GE Healthcare Biosciences (http://www.ettandige.com).5 The system is based on the binding of one of three fluorescent dyes prior to 2D differential display and enables multiplexing of up to 3 separate protein mixtures on the same 2D gel. In conjunction with appropriate image analysis software this permits automated detection, background subtraction, quantification and intergel matching. In the case of S. mutans, this has led to a more extensive appreciation of the phenotypic changes occurring during the planktonic to biofilm transition than has previously been possible using a conventional 2D proteome approach.
Material and Methods Bacterial Strain, Medium, and Growth Conditions. Cells from quadruplicate continuous cultures of S. mutans LT117 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 on modified defined mucin medium (DMM) under anaerobic conditions (95% N2/5% CO2)4 were used as an inoculum for biofilm-grown cells as well as for supplying control planktonic cultures for 2D fluorescence difference gel electrophoresis (DIGE) analyses. To obtain biofilm-grown cells, a previously described temperature controlled flow reactor with a working volume of 80 mL was used, except that the two saliva-conditioned glass slides were replaced by 4 saliva-conditioned hydroxyapatite (HA) disks (2.5 cm diameter).4 Pairs of these disks were attached with silicon (Selleys, Padstow, NSW, Australia) to cotton thread at one point on their edge, and medium at pH 7.0 was regulated to flow over the HA surfaces at a rate of 1.75 mm min-1 to mimic the flow of saliva over oral tissues.8 A number of these flow reactors, maintained at 37 °C for 48 h, were used in any one experiment in order to obtain sufficient biofilm-grown cells for DIGE analyses.4 Preparation of Saliva-Conditioned HA Disks. Since it is recognized that surface roughness plays a role in the nature and physiology of biofilm formation,9,10 HA disks were manufactured with a similar roughness to that of dental enamel, which was determined by profilometry (Department of Ceramic Engineering, The University of NSW, Sydney, NSW, Australia). By manufacturing the HA disks, the degree of porosity could also be controlled and minimized. Each HA disk was used only once and then discarded. Sterile disks were conditioned at 37 °C for 90 min with mixed filter sterilized saliva obtained from four male nonsmoking volunteers as previously described.4 Saliva-conditioned HA disks were inserted into the flow reactors immediately prior to inoculation with S. mutans cells that had been grown to steady state in a chemostat. Preparation of Cellular Proteins. Both steady state planktonic and mature 48 h biofilm cells were harvested and washed in the presence of 50 µg chloramphenicol mL-1 and 2.5 µg Sigma proteinase inhibitor cocktail mL-1 (Sigma-Aldrich, Steinheim, Germany) and lyophilized as previously described.4 Individual sets of planktonic and matching biofilm grown cells were processed simultaneously to ensure consistency. Each set of cells was prepared separately to monitor reproducibility of 2162
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the preparation method. A total of 4 sets of cells derived from four separate chemostats were prepared in this manner. Aliquots of 20 mg of lyophilized planktonic or biofilm cells were partitioned into ‘wall’ and ‘cellular’ fractions using a modification of the method described by Jonquie`res et al.11 and then recombined as a ‘whole cell’ fraction prior to DIGE analysis. In essence, each aliquot of cells was resuspended in 1 mL of 10 mM potassium phosphate buffer pH 6.5 containing 1 mM CaCl2, 50 µg Sigma proteinase inhibitor cocktail mL-1 and 1000 U mutanolysin mL-1 (Sigma-Aldrich). The cell suspension was resuspended by gentle sonication on ice at 130 W for 2 × 10 s using a cup horn (Branson Sonifer 450; Branson Ultrasonics Corporation, Danbury, CT), incubated at 37 °C for 90 min and then at 50 °C for 30 min to hydrolyze the S. mutans cell wall. The muramidase-treated cells were cooled on ice and re-sonicated (130 W, 2 × 10 s) prior to the addition of 5 µL of a solution containing 25 µg of both RNase A and DNase I (Boehringer Mannheim, Sydney, NSW, Australia). The cells were then dialyzed against 2 × 4 L of 18 MΩ H2O at 4 °C for a total of 4 h. To enhance ‘wall’ removal, the dialyzed samples were frozen overnight (16 h) at -20 °C, thawed on ice and resonicated (130 W, 3 × 10 s). Solubilized ‘wall’ proteins were separated from the remaining ‘cellular’ material by centrifugation (15 000 × g, 10 min, 4 °C) and the ‘cellular’ material resuspended by sonication (130 W, 4 × 10 s) on ice in 50 mM Tris pH 7.2 prior to centrifugation (15 000 × g, 10 min, 4 °C). The washed ‘cellular’ material was then stored at -20 °C and the two supernatant fractions containing ‘wall’ proteins precipitated with 25% (w/v) TCA by incubation at -20 °C for 16 h. The precipitated ‘wall’ proteins were then recovered by centrifugation (12 000 × g, 15 min, 4 °C), washed first with icecold 2-propanol and then methanol (12 000 × g, 15 min, 4 °C) before being dried in air and stored at -20 °C until required. Proteins from the ‘cellular’ fraction were extracted with the Amersham 2D Clean-Up Kit (GE Healthcare Biosciences Australia, Sydney, NSW, Australia) according to the manufacturer’s recommendation. ‘Cellular’ and ‘wall’ proteins were then resuspended in a rehydration solution consisting of 30 mM Tris pH 8.6 containing 7 M urea, 2 M thiourea, and 4% (w/v) CHAPS. The protein fractions were subsequently recombined and the final protein concentration adjusted to 50 mg mL-1 with rehydration solution after quantifying the protein concentration with Amersham PlusOne 2D Quant Kit (GE Healthcare Biosciences Australia). The pH of the sample was then adjusted to pH 8.5-8.7 on ice in preparation for CyDye labeling. By fractionating, and subsequent recombining the ‘cellular’ and ‘wall’ fractions, a significant increase in the number of protein spots were observed on 2D gels compared with previous whole cell preparation methods.12 CyDye Labeling of Proteins. Proteins were labeled with CyDye DIGE fluors (GE Healthcare Biosciences Australia) according to the manufacturer’s protocols. In brief, 50 µg of protein from each of the quadruplicate steady state planktonic and mature biofilm grown cells were minimally labeled with 400 pmol of either the Cy3 or Cy5 DIGE fluors. Samples of each cell type (planktonic or biofilm) were alternately labeled with Cy3 and Cy5, thus accounting for possible intensity differences between the dyes and avoiding bias which may result from differential labeling by just one dye (Figure 1). A pooled set of internal standards comprising 50 µg aliquots from each of the 8 samples (total 400 µg) was also minimally labeled
Analysis of Streptococcus mutans Biofilms
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Figure 1. 2D DIGE protocol and scans resulting from the CyDye labeling procedure. Each gel contains a mixture of a matched set of planktonic and biofilm protein samples with an equal aliquot of pooled standard. Pooled standards were always labeled with Cy2 (orange outline) while the planktonic and biofilm samples are alternately labeled with either Cy3 (blue outline) or Cy5 (pink outline).
with Cy2 DIGE fluors. Labeling was performed on ice for 30 min in the dark and quenched with 1 mM lysine for 10 min on ice. DIGE. Prior to the first dimension separation of proteins, IPG strips (18 cm, pH 4-7 and 3-11; GE Healthcare Biosciences Australia) were rehydrated overnight with DeStreak Rehydration Solution (GE Healthcare Biosciences Australia) containing 2% (v/v) of the appropriate IPG buffer (GE Healthcare Biosciences Australia). Equal aliquots of matching pairs of labeled protein samples from planktonic and biofilm grown cells along with the internal protein standard were combined to produce 4 sample sets each containing a Cy2/Cy3/Cy5-labeled mixture with a total of 150 µg of protein (Figure 1). DeStreak Rehydration Solution (100 µL) containing 2% (v/v) IPG buffers and 5 mM tributylphosphine (Sigma-Aldrich) was added to each set of samples and allowed to stand at room temperature (20-22 °C) for 30 min. Freshly prepared iodoacetamide was then added to a final concentration of 10 mM and the samples incubated for a further 30 min at room temperature. Samples were then cup-loaded onto the IPG strips and focused at 20 °C on a Multiphor II (GE Healthcare Biosciences Australia) for a total of 70 kVh for the pH 4-7 IPG strips and 65 kVh for the pH 3-11 IPG strips. Focused IPG strips were then equilibrated as described previously,4 before separation of proteins in the second dimension on 12.5% (w/v) acrylamide gels using an Ettan DALTsix electrophoresis unit with low fluorescent glass plates (GE Healthcare Biosciences Australia). All incubation and electrophoresis procedures were carried out in the dark.
Preparative 2D displays of proteins from planktonic and biofilm grown cells were prepared separately but in parallel with the DIGE experiment. For the first dimension separation on pH 4-7 IPG strips, 1.5 mg of each protein sample was focused for a total of 83 kVh after loading onto the strips using the paper bridge method.13 For the pH 3-11 IPG strips, 0.9 mg of protein was passively rehydrated overnight into the IPG strips and focused for a total of 78 kVh. Image Analysis. DIGE gels were scanned with a Typhoon 9410 scanner (GE Healthcare Biosciences Australia) producing a Cy2, Cy3, and Cy5 image for each of the four gels at excitation wavelengths of 488, 532, and 633 nm and emission wavelengths of 520, 580, and 670 nm, respectively. After CyDye imaging, gels were removed from the low fluorescent glass plates and fixed and stained with SyproRuby (Invitrogen Australia Pty Ltd, Mount Waverley, VIC, Australia). SyproRuby stained images were also acquired at an excitation wavelength of 532 nm with a 610BP30 emission filter, before the gels were ‘double stained’ and visualized with Coomassie Blue G-250 (Sigma-Aldrich).12 Preparative gels were similarly fixed and stained with SyproRuby prior to visualization with Coomassie Blue G-250. Image analysis was carried out with DeCyder 5.02 software (GE Healthcare Biosciences Australia) using the batch processor facility. The batch processor automatically analyzed the scanned images first using the Differential In-gel Analysis (DIA) module where the Cy2, Cy3, and Cy5 image from each gel was normalized and the Cy3 and Cy5 samples compared with the Cy2 internal standard. This paired ratio data was then utilized Journal of Proteome Research • Vol. 4, No. 6, 2005 2163
research articles in the Biological Variation Analysis (BVA) module where the Cy2 internal standard images from the quadruplicate gels were matched, allowing the spot ratios between all 4 gels to be compared. A list was thus generated containing the protein spot ratio and T-test value for each spot. Relevant spots were selected by narrowing the filter settings such that differences in the spot volume between the proteins from planktonic and biofilm grown cells were g 1.5 with a T-test confidence g 95% (P e 0.05). Protein Identification. Individual protein spots, excised in gel pieces from the preparative 2D gels, were washed in 150 µL of 18 MΩ H2O and then destained by washing 3 × in 20 mM NH4HCO3 in 50% (v/v) acetonitrile in a 96-well microtiter plate at room temperature using a multichannel pipet. The dried gel pieces were then rehydrated in 2 µL of 20 mM ammonium bicarbonate containing 0.004% (w/v) trypsin at 4 °C for 60 min. When re-swollen, 5-10 µL of 18 MΩ H2O was added to each gel piece and the samples incubated for 16 h at 37 °C. An additional 1.0 µL of trypsin solution was added to each sample 1 h before the end of the 37 °C incubation. Four microliters of each digested sample were then layered in 1µL aliquots onto a matrix-assisted laser desorption ionization (MALDI) 20 × 20 sample plate with hydrophobic mask (Micromass, Manchester, UK). Each aliquot was dried between applications and 400 nL of matrix (10 mg R-cyano-4-hydroxycinnamic acid in 70% (v/v) acetonitrile mL-1 containing 1% (v/v) trifluoroacetic acid) was placed over each spot. MALDI-time-of-flight (TOF) MS was performed on a VoyagerDE PRO MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, MA). Peak lists were generated using Data Explorer (PerSeptive Biosystems) calibrated against trypsin autolysis peaks (m/z 842.51, 2211.10). Putative proteins were identified using the General Protein Mass Analysis for Windows (GPMAW; Lighthouse Data, Odense, Denmark) with a mass tolerance setting of 45 indicated identity or extensive homology and a score >23 indicated significant homology. Protein Nomenclature. Protein identifications 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 alternative forms of a protein including isoforms are given by an alphabetized suffix. The term ‘isoform’ is used to describe the multiple charged forms of a protein that exist on a given 2D gel, where 2164
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the observed Mr for each form calculated from the 2nd (SDSPAGE) dimension deviated by approximately 10% or less, and where there was no evidence from peptide-mass mapping of some form of truncation or degradation. The observed Mr and pI coordinates on DIGE gels were calculated using the DeCyder software based on the Mr and pI of randomly selected landmark proteins predicted from their gene sequence. Landmark proteins (12-14 per IPG range) were chosen to represent as wide a distribution of coordinates on each 2D gel as possible.
Results and Discussion Analysis of DIGE Displays. Protein spots that displayed statistically significant differences g 1.5-fold (P < 0.05) in their level of expression between planktonic and biofilm grown cells, and that were visually confirmed not to be artifacts, were the primary focus for identification by MS. On the basis of these criteria, a total of 132 differentially expressed protein spots were detected on pH 4-7 and pH 3-11 range gels. One-hundred sixteen of these protein spots, representing 73 different proteins, were identified by MS. Of these 73 proteins, 64 were down-regulated in biofilm grown cells. (Figure 2, Table 1) This result compares with a total of 34 proteins that were identified using traditional 2D analyses as being altered in a statistically significant manner or that were unique to one growth state or the other.4 Of these 34 proteins, 16 were common to both studies. DIGE analysis failed to identify any protein that was uniquely expressed in either planktonic or biofilm grown cells although 7 such proteins and 13 forms of 6 other proteins were found using traditional 2D analyses.4 Several explanations could account for these discrepancies. First, the very high sensitivity of Cy-Dyes compared with SyproRuby allowed low levels of proteins to be observed in the DIGE analyses, negating the likelihood of unique status in planktonic or biofilm grown cells. Second, the use of a series of narrow range IPG strips, led to better protein spot separation in the traditional 2D analyses.4 Despite these observations, only one identified protein common to both studies showed a contradictory trend in its level of expression in biofilm grown cells. This protein, acetoin dehydrogenase, ButA, forms part of a pathway giving rise to an alternative fermentation product other than lactate. ButA was up-regulated 3.4-fold in biofilms grown on saliva-conditioned glass slides,4 but down-regulated 2.4-fold on similarly conditioned HA surfaces. Whether this was due to a change in the surface on which the biofilm was grown remains to be determined, as it is known that alterations in surface properties can alter the biofilm phenotype.16,17 While many of the differentially expressed protein spots were identified on both the pH 4-7 and pH 3-11 2D gels, 7 different protein spots (including 3 putatively degraded forms) were unique to the pH 4-7 gels and 12 protein spots representing 5 different proteins were unique to the pH 3-11 2D gels (Table 1). Among the identified protein spots were 35 that were found to be expressed in a significantly different manner (P < 0.05) in biofilm grown cells only when analyzed on pH 4-7 2D gels (Table 1). The most plausible explanation for this observation was the better resolution of protein spots in gels of this pH range. Supporting this suggestion was the observation that 6 protein spots that were merged on pH 3-11 2-D gels were resolved on pH 4-7 gels. Regardless of the reason for this statistical discrepancy, comparable values were generally observed for the differential ratios of protein spots identified on both the pH 4-7 and pH 3-11 2D gels (Table 1). Ten
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Analysis of Streptococcus mutans Biofilms Table 1. Proteins Differentially Expressed in S. mutans Biofilms protein ratio planktonicb protein name, definition, EC no.
spot IDa
pI 3-11d
pI 4-7e
biofilmc
2DE coordinates (Mr/pI) theoreticalf
pI pI 3-11 4-7
observedg pI 3-11
pI 4-7
I-Metabolism 1-Central Metabolism Smut0493ho 2.11 Smut0277 1.75 Smut1090-ahi 1.60 Smut1090-bh 1.60 Smut0088-ao 1.41 Smut0088-b 1.87 Smut0088-co 2.16 Smut0652-ajo 1.70 Smut0652-bj 2.10 Smut0652-clo 1.25 Smut0325-a 2.47
3.12 2.15 2.18 2.13 2.27 2.20 2.32 2.39 2.32 2.04 2.74
33621/4.7 49410/4.7 35758/5.5 35758/5.5 31412/4.8 31412/4.8 31412/4.8 27197/4.9 27197/4.9 27197/4.9 36055/6.0
37000/4.5 49000/4.4 37500/5.3 37500/5.5 30000/4.6 30000/4.7 30000/4.8 26000/4.6 26000/4.7 23000/4.4 40500/5.4
Smut0325-b-ck
2.50
2.55/ 3.85
36055/6.0
Smut0325-d
2.11
2.44
36055/6.0
40500/5.6 39500/5.6, 40000/5.5 40500/5.8 40000/5.8
Smut0325-e
2.08
2.48
36055/6.0
40500/6.0 40500/6.0
Smut0325-f
2.33
3.05
36055/6.0
41000/6.3 40500/6.1
Smut0325-g
2.70
3.22
36055/6.0
41000/6.6 40500/6.3
Smut0326-ao Smut0326-bo Smut0326-c Smut0326-d Smut0326-el Smut0326-fl Smut0326-ghil Smut0326-hlo Smut0326-il Smut0326-jl Smut0618-aho
2.99 2.96 2.07 3.08 2.79 2.97 4.50 4.00 2.23 2.04 2.36 1.68 2.27 1.74 1.86 1.86 2.15 1.52 1.88 1.39 1.90
42033/5.1 42033/5.1 42033/5.1 42033/5.1 42033/5.1 42033/5.1 42033/5.1 42033/5.1 42033/5.1 42033/5.1 51165/4.9
42500/4.8 42500/5.0 42500/5.1 42500/5.3
Smut0618-bh
1.87
51165/4.9
40500/5.2 40000/5.2
1.70 2.39 Smut0543-bj 2.10 2.32 Smut0543-c 2.14 2.53 Smut1140-a-bkmo 1.24 1.87/ 1.91
26035/5.0 26035/5.0 26035/5.0 46846/4.5
26000/4.6 25500/4.7 26000/4.9 46846/4.5
Smut1018-ao Smut1018-b Smut1207-a Smut1207-b Smut0131-a-bko
1.80 2.46 2.49 2.31 1.51 1.97 1.79 2.44 3.52 3.36/ 3.11
35231/4.9 35231/4.9 26889/6.4 26889/6.4 96988/5.5
37500/4.8 38000/4.9 30000/6.0 30000/6.4 68500/5.2
AdhE, alcohol-acetaldehyde dehydrogenase, 1.2.1.10 Smut0131-chilo 1.37 1.69 AdhE, alcohol-acetaldehyde dehydrogenase, 1.2.1.10 Smut0131-dhl 3.09 3.59 Pta, phosphotransacetylase, 2.3.1.8 Smut0952 2.64 3.35 TktA, transketolase, 2.2.1.1 Smut0262mo 1.71 2.17 RpiA, ribose 5-phosphate isomerase, 5.3.1.6 Smut1129 1.62 MsmK-like protein, ABC-type transport system Smut1428m 2.00 2.71 ATP-binding protein (maltose) GlgD, glycogen biosynthesis protein, 2.7.7.27 Smut1398 2.54 4.35 GlgC, glucose-1-phosphate adenylyltransferase, Smut1399h 2.86 4.96 2.7.7.27 FabK, trans-2-enoyl-ACP reductase II Smut1588-ao 1.71 1.91 FabK, trans-2-enoyl-ACP reductase II Smut1588-bl 1.66 GabD, NAD-dependent aldehyde dehydrogenase Smut1930-a-bjk 1.99 4.06/ 3.55 (possible succinic semialdehyde dehydrogenase), 1.2.1.16 Nrd, nitroreductase family protein (possible NADH Smut1135 1.57 1.97 dehydrogenase) PpaC, manganese-dependent inorganic Smut1537mo 1.06 2.11 pyrophosphatase (type II PPase), 3.6.1.1 2-Phosphotransferase System (PTS) PtsH, HPr Smut0616-ahi 2.71 3.22 PtsH, HPr Smut0616-bhin 2.35 2.87 ManL, mannose-specific component IIAB, 2.7.1.69 Smut1707 3.70 2.97
96988/5.5 96988/5.5 36369/4.8 71067/4.8 24554/4.6 42314/6.4
23500/6.3 18000/6.9 35500/4.6 71067/4.8
42323/6.0 42189/4.6
44500/6.0 44000/5.9 45500/4.4 44500/4.5
33522/5.9 33522/5.9 50448/4.8 22665/5.2
34000/6.0 34000/6.0 24000/7.5 50500/4.6 49500/4.8, 50000/4.8 24000/5.3 24000/5.4
33389/4.6
33389/4.6 33389/4.6
8919/4.7 8919/4.7 35549/5.0
12500/4.3 12000/4.5 12500/4.6 12000/4.8 38000/5.0 38000/5.1
Glk, glucose kinase, 2.7.1.2 Gpi, glucose-6-phosphate isomerase, 5.3.1.9 PfkA, 6-phosphofructokinase, 2.7.1.11 PfkA, 6-phosphofructokinase, 2.7.1.11 FbaA, fructose-1,6-biphosphate aldolase, 4.1.2.13 FbaA, fructose-1,6-biphosphate aldolase, 4.1.2.13 FbaA, fructose-1,6-biphosphate aldolase, 4.1.2.13 TpiA, triosephosphate isomerase, 5.3.1.1 TpiA, triosephosphate isomerase, 5.3.1.1 TpiA, triosephosphate isomerase, 5.3.1.1 GapC, glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.12 GapC, glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.12 GapC, glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.12 GapC, glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.12 GapC, glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.12 GapC, glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.12 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 Pgk, phosphoglycerate kinase, 2.7.2.3 GapN, NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.9 GapN, NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, 1.2.1.9 PmgY, phosphoglyceromutase, 5.4.2.1 PmgY, phosphoglyceromutase, 5.4.2.1 PmgY, phosphoglyceromutase, 5.4.2.1 Eno, enolase, 4.2.1.11 Ldh, L-lactate dehydrogenase, 1.1.1.27 Ldh, L-lactate dehydrogenase, 1.1.1.27 ButA, acetoin reductase, 1.1.1.5 ButA, acetoin reductase, 1.1.1.5 AdhE, alcohol-acetaldehyde dehydrogenase, 1.2.1.10
1.97
Smut0543-ajo
34000/5.5 33500/5.8 28500/6.0 28500/6.3 28500/6.7 40500/5.0
42314/6.4
36000/4.7 47000/4.6 37500/5.4 37000/5.5 30000/4.8 30000/4.9 30000/5.0 25500/4.8 255000/4.9 22500/4.5 40000/5.4
42000/5.0 42000/5.1 42000/5.2 42000/5.3 33500/5.4 33500/5.5 33500/5.7 28500/5.9 28500/6.2 29000/6.4 40500/5.1
25500/4.8 25500/4.9 26000/5.0 46846/4.5, 46846/4.5 37500/4.9 37500/5.0 29500/5.9 30000/6.2 67000/5.4, 67500/5.3 23500/6.2 18500/6.5 36000/4.8 71067/4.8 25000/4.5 42314/6.4
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Table 1. (Continued) protein ratio planktonicb protein name, definition, EC no.
spot IDa
pI 3-11d
pI 4-7e
2DE coordinates (Mr/pI)
biofilmc pI 3-11
theoreticalf
pI 4-7
3-Amino-Acid and Co-factor Biosynthesis and Salvage Pathways DapD, tetrahydrodipicolinate succinylase, 2.3.1.117 Smut0285o 2.42 2.48 2.09 2.01 IlvC, ketol-acid reductoisomerase, 1.1.1.86 Smut0211mo IlvD, dihydroxy-acid dehydratase, 4.2.1.9 Smut1931-ao 1.56 2.24 IlvD, dihydroxy-acid dehydratase, 4.2.1.9 Smut1931-b 2.10 2.43 IlvE, branched-chain amino acid aminotransferase, Smut1099o 1.59 2.00 2.6.1.42 GdhA, NADP-specific glutamate dehydrogenase, 1.4.1.4 Smut0829-ao 1.21 1.92 GdhA, NADP-specific glutamate dehydrogenase, 1.4.1.4 Smut0829-bo 1.40 1.80 GdhA, NADP-specific glutamate dehydrogenase, 1.4.1.4 Smut0829-c 1.89 2.09 ProC, pyrroline carboxylate reductase, 1.5.1.2 Smut1792 1.57 2.15 SerC, phosphoserine aminotransferase, 2.6.1.52 Smut1508 2.01 2.33 SerA, D-3-phosphoglycerate dehydrogenase, 1.1.1.95 Smut1505 2.45 2.88 CysK, cysteine synthetase A, 2.5.1.47 Smut0449 1.56 1.29 PdxK, pyridoxal kinase, 2.7.1.35 Smut0866o 1.37 1.71 ThiD, phosphomethylpyrimidine kinase, 2.7.4.7 Smut0077 1.60 1.86 MetK, S-adenosylmethionine synthetase, 2.5.1.6 Smut1430o 1.57 2.62 PepD, dipeptidase, 3.4.13.18 Smut1190jko 1.99 4.06 LivF, branched chain amino acid ABC transporter, Smut1517 1.55 1.85 ATP-binding protein AtmD, ABC transporter, ATP-binding protein Smut1003j 1.94 1.54 2.38 2.99 GlnQ, glutamine ABC transport, ATP-binding protein Smut1380him 4-Nucleotide Biosynthesis and Salvage Pathways GuaB, inosine monophosphate dehydrogenase, 1.1.1.205 Smut1957-a 2.61 2.27 GuaB, inosine monophosphate dehydrogenase, 1.1.1.205 Smut1957-bm 2.95 3.71 GuaB, inosine monophosphate dehydrogenase, 1.1.1.205 Smut1957-c 3.11 3.24 PyrH, uridylate kinase (UMP-kinase), 2.7.4.Smut1479 2.33 Upp, uracil phosphoribosyltransferase, 2.4.2.9 Smut1525 2.16 2.49 Adk, adenylate kinase (ATP-AMP transphosphorylase) Smut1821o 2.43 1.81 (superoxide-inducible protein 16) (SOI16), 2.7.4.3 5-Cell Wall Biosynthesis PgmA, phosphoglucomutase, 5.4.2.2 Smut0985-a-bk 3.58 4.36/ 4.62
observedg pI 3-11
pI 4-7
24092/4.5 37273/4.9 60772/5.1 60772/5.1 37718/4.9
25000/4.2 37273/4.9 56000/4.9 56000/5.0 42000/4.7
24500/4.4 37273/4.9 55000/5.1 55000/5.2 41500/4.9
48221/5.3 48221/5.3 48221/5.3 27087/5.2 40352/5.0 42785/5.6 32423/6.1 31607/6.5 27669/6.1 43116/5.2 53026/4.8 25577/6.0
44500/5.3 44500/5.5 44500/5.7 25500/5.2 45000/5.0 41500/5.9 23500/6.7 28000/6.6 28000/5.9 52500/5.2 50500/4.6 25000/6.2
43500/5.3 43500/5.5 44000/5.7 25000/5.3 45000/5.1 41000/5.8 24000/6.4 28500/6.3 27500/5.8 52000/5.3 49500/4.8 25000/6.1
27057/6.6 28939/6.2
27000/6.7 27000/6.7 28939/6.2 28939/6.2
53084/5.9 53084/5.9 53084/5.9 26317/5.4 23036/6.7 23623/5.1
54500/5.9 53000/5.7 53084/5.9 53084/5.9 54000/6.2 54000/6.0 26500/5.5 25500/6.9 26000/6.6 26000/5.1 26000/5.2
63086/4.7
RmlA, glucose-1-phosphate thymidyltransferase, 2.7.7.24 Smut1330-a 1.61 2.52 RmlA, glucose-1-phosphate thymidyltransferase, 2.7.7.24 Smut1330-b 2.03 2.79 6-Inorganic Ion Transport SloA, ABC transporter (iron and/or manganese), Smut0164jm 1.94 1.54 ATP-binding protein YurY homolog, ABC transporter, ATP-binding protein Smut0224o 1.56 1.73 (potential iron regulation) II-Cellular Processes 1-Cell Division FtsZ, cell division protein Smut0502-ao 2.18 3.02 FtsZ, cell division protein Smut0502-bl 1.60 2.69 2-Translation Sys, seryl-tRNA synthetase, 6.1.1.11 Smut1714 2.89 1.73 RplJ, 50S ribosomal protein L10 Smut0869-ao 1.50 2.05 RplJ, 50S ribosomal protein L10 Smut0869-bl 2.00 2.59 1.83 2.02 Rpl, 50S ribosomal protein L7/L12 Smut0872o klo EF-Tu, translation elongation factor Smut0651a-b 1.59 1.78/ 2.47
32281/4.7 32281/4.7
57500/4.4 55000/4.6, 55000/4.6 31500/4.5 31500/4.6 31500/4.6 31500/4.7
26778/6.7
26778/6.7 26778/6.7
28267/4.4
30000/4.2 29000/4.3
45732/4.2 45732/4.2
52000/4.0 50500/4.1 50500/4.1 49000/4.2
48381/4.9 17651/4.7 17651/4.7 12396/4.3 43907/4.7
50000/4.8 16000/4.5 12500/4.5 11500/4.0 29000/4.4
EF-P, translation elongation factor P EF-TS, translation elongation factor Ts Frr, ribosome recycling factor
20765/4.7 37707/4.7 20627/5.6
Smf, DNA processing protein (Smf family) SsuRB, type II restriction endonuclease SsbA, single-stranded DNA-binding protein SsbA, single-stranded DNA-binding protein RecA, recombinase A protein RecA, recombinase A protein RecA, recombinase A protein RecA, recombinase A protein RecA, recombinase A protein RecA, recombinase A protein RecA, recombinase A protein RecA, recombinase A protein Smut0697, conserved hypothetical protein Smut0697, conserved hypothetical protein Smut0760, hypothetical protein
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Journal of Proteome Research • Vol. 4, No. 6, 2005
Smut1682 1.62 1.69 Smut1846o 1.44 1.91 Smut1478 1.61 2.50 3-Quorum Sensing Regulated Smut0911m Smut0459 Smut1785-ai Smut1785-bin Smut1892-a Smut1892-b Smut1892-c Smut1892-dl Smut1892-el Smut1892-fhl Smut1892-ghil Smut1892-hl Smut0697-ai Smut0697-binm Smut0760hio
4.73 31318/9.5 7.38 6.18 36375/6.8 17.38 17.09 14826/6.6 40.41 50.34 14826/6.6 3.84 41389/5.1 28.84 41389/5.1 8.45 41389/5.1 11.31 41389/5.1 4.97 41389/5.1 6.58 4.10 41389/5.1 1.87 41389/5.1 3.96 41389/5.1 4.41 8107/4.9 13.46 16.09 8107/4.9 5.75 6.76 60276/6.8
48500/4.8 16500/4.7 125000/4.6 11500/4.1 28500/4.5, 28500/4.6 25000/4.5 25000/4.7 43500/4.6 42000/4.7 24000/5.8 24000/5.8 31318/9.5 34000/7.2 14500/6.4 14500/6.9 37500/8.2 38000/8.7 38000/8.8 36000/7.9 36000/8.2 30500/7.0 30500/8.5 29000/7.3
33500/6.7 14500/6.2 15000/6.5
31000/6.6
8000/4.7 8107/4.9 8107/4.9 56440/6.7 55969/6.4
research articles
Analysis of Streptococcus mutans Biofilms Table 1. (Continued) protein ratio planktonicb protein name, definition, EC no.
GtfC, glucosyltransferase-SI, 2.4.1.5 GtfC, glucosyltransferase-SI, 2.4.1.5 CppA, C3-degrading proteinase Smut0079, conserved hypothetical protein Smut0139, conserved hypothetical protein Smut0188, hypothetical protein Smut0710, conserved hypothetical protein Smut0997, conserved hypothetical protein Smut1255, conserved hypothetical protein Smut1255, conserved hypothetical protein MccF, conserved hypothetical protein (possible immunity protein) Smut1887, conserved hypothetical protein
spot IDa
pI 3-11d
pI 4-7e
III-Virulence Factors Smut0915-ah Smut0915-bhl Smut0360o 1.51 1.89 IV-Unknown Function Smut0079 1.22 1.52 Smut0139 2.01 Smut0188im Smut0710i 3.01 Smut0997m 2.17 2.58 Smut1255-a 1.57 1.91 Smut1255-b 1.78 2.29 Smut1849 2.51 Smut1887i
a
1.75 b
2DE coordinates (Mr/pI)
biofilmc pI 3-11
observedg
pI 4-7
6.00 4.36
1.79
theoreticalf
2.89
pI 3-11
pI 4-7
162970/10.0 162970/10.0 28983/4.6
74186/8.9 70341/8.8 27050/4.4
26313/4.6
20341/6.5 28136/4.5 15778/5.0 12872/4.4 22469/5.4 29425/5.4 29425/5.4 36093/6.9
24555/6.7 15778/5.0 22469/5.4 28460/5.3 28598/5.5 37408/7.2
10418/4.8
25229/6.4 26872/4.5 15778/5.0 9919/4.3 22469/5.4 28398/5.3 28249/5.5 9046/4.5
c
Based on gene ID from the Oralgen website (http://www.oralgen.lanl.gov/). Fold increase occurring during planktonic growth. Fold increase occurring during biofilm growth. d Data from DIGE analysis of matched gels with a pH range of 3-11. e Data from DIGE analysis of matched gels with a pH range of 4-7. f Data obtained from the Oralgen website (http://www.oralgen.lanl.gov/). g Assigned M and pI based on the coordinates of landmark proteins. h Protein match r falls below 20% coverage. i Protein match falls below 5 peptides. j Two protein IDs for one spot (Smut0652-aj and Smut0543-aj; Smut0652-bj and Smut0543-bj; jk and Smut1190jk; Smut1003j and Smut0164jm). k Merged proteins (ie two protein spots on pH 4-7 range gels, but only one protein spot on pH Smut1930-a-b 3-11 range gels). l Truncated protein based on the observed Mr being 0.05).
hypothetical proteins were altered in their level of expression by growth of S. mutans in a biofilm (Table 1). Stress-Responsive Proteins. In some bacterial communities, biofilm formation has been observed to be triggered by stress factors,18-20 while a number of studies have identified the importance of stress-responsive proteins in the development of biofilms, though usually these are restricted to studies with isogenic mutants subjected to various stress conditions.3,19-21 Multiple stress transcripts have also been identified in biofilm cells of Escherichia coli using microarray analyses to evaluate global gene expression.22,23 Such studies have led to the suggestion that a stress response may be a distinct phenotypic feature of bacterial biofilms.3,24 However, it is now becoming evident that the expression of stress-responsive proteins in biofilms is not necessarily a requirement for biofilm formation per se, but rather a result of an imposed environmental stress on the biofilm similar to the situation when planktonic cells are subjected to stress.4,25 DIGE analysis of S. mutans grown as a biofilm in a neutral pH environment failed to show any change in the level of stress-responsive proteins, including molecular chaperones and protective replicative and transcriptional activities that were elevated in planktonic cells grown under acidic conditions.26 In fact, of the proteins belonging to these groups, the proteinases, PepD and CppA, were down-regulated by an average of 3.0- and 1.7-fold, respectively, in biofilm grown cells (Table 1). These results were consistent with previous findings using traditional 2D analysis.4 Proteins Up-Regulated in Biofilm Grown Cells. Among the 9 proteins that were up-regulated in biofilm grown cells, was a hypothetical protein, Smut0188, of unknown function which was up-regulated 2.3-fold (Table 1). The gene encoding this protein did not possess any obvious features which allowed it to be characterized further. The relevance of the changes in the level of expression of the remaining 8 proteins are discussed below. (i) Glucan Synthesis. Sucrose is the most cariogenic carbohydrate known, partly due to its conversion into extracellular
polysaccharides by high Mr bacterial glucosyltransferases (Gtf) that synthesize glucans (dextrans and mutans) that form an integral part of dental plaque. One of the three Gtf of S. mutans, GtfC, along with a truncated form of the same protein were up-regulated by a factor of 6.0- and 4.4-fold, respectively, in biofilm grown cells (Table 1). GtfC has an essential role in sucrose-dependent attachment of S. mutans to surfaces in mono-culture.27 In S. mutans, the gtfC gene forms an operon with gtfB with the operon having two promoters. The promoter upstream of the gtfB/gtfC genes, PgtfB, can produce mRNA containing both gtfB and gtfC.28 However, the second promoter, PgtfC, that lies between the two genes upstream of gtfC is an order of magnitude stronger than PgtfB,29,30 a fact which may account for why GtfC but no other Gtf was visualized on the 2D gels. Recent evidence suggests that the autoinducer-2 (AI-2; furanosyl borate diester) quorum sensing system plays a role in Gtf expression, since gtfB and gtfC, but not the gtfD, are upregulated in an autoinducer synthase, luxS, mutant in the presence of sucrose when compared with wild-type cells. Furthermore, biofilm formation by the luxS mutant in the presence of sucrose is markedly reduced compared with the wild type.31 Superimposed on top of these findings is the observation that the VicRK signal transduction system acts as a positive regulator of gtfB, gtfC, and gtfD as well as the fructosyltransferase gene, ftf (the product of which produces fructan from sucrose), and the glucan-binding protein gene, gbpB. This twocomponent signal transduction system also influences transformation efficiency and biofilm formation in vitro even though the signal molecule for this system is currently unknown.32 Whether the gtf genes are subject to other sensor/regulator control is unknown. However, despite the -10 promoter sequences of PgtfB (TACAATT) and PgtfC (TACAATA) of the gtfB and gtfC genes being similar to the competence induced (cin)box consensus sequence, TACGAATA, to which the alternative sigma factor ComX (σx) binds to initiate the transcription of late competence genes,33 it is unlikely that these genes are Journal of Proteome Research • Vol. 4, No. 6, 2005 2167
research articles
Rathsam et al.
Figure 2. 2D DIGE scans of S. mutans protein samples. (A) pH range 3-11. (B) pH range 4-7. Spot ID numbers correspond to the gene identification for the S. mutans UA159 genome on the Oralgen web site (http://www.oralgen.lanl.gov/).44 Protein spots identified with yellow numbers are up-regulated in planktonic cells, while those labeled with pink numbers are up-regulated in biofilms. Boxed regions on (A) correspond to the expanded spot profiles of (C) which show the section of the 2D DIGE scan, the corresponding 3D views and a graphical view of the normalized log abundance of each protein spot relative to the standard for each of the quadruplicate gels (dots). The crosses on the graphical views correspond to the average of the normalized log abundance for each growth condition. 2168
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Analysis of Streptococcus mutans Biofilms
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Figure 3. Putative cin-box promoters. Each promoter contains a conserved cin-box sequence, ‘taCGAATA’ (red), a conserved ‘T’ at -25, a conserved ‘T’ -35 and several ‘T’ closely preceding the -10 cin-box.34,47 The sequence in bold represents a putative stem loop structure surrounding the putative cin-box of the SMu0760 gene. Upstream of the start codon (green ATG) of each gene is a conserved Shine-Dalgarno sequence (SD; blue) which is complementary to the 3′ end of 16S rRNA (data not shown for recA and ssuRB genes).
under the control of the competence quorum sensing system since the missing G means that these sequences are not likely to be functional cin-boxes.34 (ii) Iron-sulfur Proteins. Cysteine synthetase A (O-acetylserine sulfhydrylase), CysK, was up-regulated 1.6-fold on pH 3-11 gels. CysK catalyzes the biosynthesis of cysteine from acetylserine and sulfide and has been reported to be involved in stress response in Listeria monocytogenes,35 E. coli,36 Lactococcus lactis,37 and Haemophilus parsuis.38 Information on cysteine biosynthesis in Gram-positive bacteria is limited, though recent studies have shown that a LysR-type regulator protein, CmbR, is essential for the expression of the metC-cysK operon in L. lactis.39 In S. mutans, the cysK gene is located differently on the chromosome to that of L. lactis. The cysK gene is downstream of SMu0450 coding for a protein of unknown function. These two genes are transcribed in the opposite direction to the late competence operon, comF-comFC which is to be found next to SMu0450 (http://www.oralgen.lanl.gov/). A cin-box-like element is to be found upstream of comF between Smu0405 and comF, but due to its orientation may not influence cysK expression. Interestingly, CysK, acting as an O-acetylserine sulfhydrylase, has been implicated in the formation of iron-sulfur ([Fe-S]) clusters in L. lactis,40 by regulating sulfide mobilization from cysteine in a manner similar to that catalyzed by O-acetylserine sulfhydrylases A and B (CysK and CysM, respectively) in E. coli.41 Over 100 different proteins have been described as containing [Fe-S] clusters.42 For instance, in S. mutans, the oxygen sensitive enzyme, pyruvate formate-lyase, Pfl (Smut0363), that contains a glycyl radical in its active structure and is the first enzyme in a pathway that converts pyruvate to acid fermentation products other than lactate, is maintained in an active state by the Pfl-activating enzyme, Act (Smut0444), that contains an [Fe-S] cluster.43 Although Act was not identified in the current study, Pfl (Smut0363) was up-regulated 2.1-fold on pH 4-7 gels and 1.8-fold on pH 3-11 gels, though not in a statistically significant manner (P < 0.059 and P < 0.069 respectively; data not shown). (iii) Expression of Competence-Related Proteins in S. mutans Biofilms. Six of the 9 proteins that were up-regulated in biofilm grown cells contained a putative cin-box in their promoter region (Table 1, Figure 3). Four of these proteins, the recombinase A protein, RecA, the single strand binding protein, SsbA, the DNA processing protein, Smf, and a conserved hypothetical protein, Smut0697 have previously been identified as being up-regulated in biofilm grown cells.4 All but the conserved hypothetical protein, Smut0697, appear to be essential for competence.33 Despite this, the gene encoding Smut0697 has now been shown to be up-regulated in biofilm
grown cells in two separate studies (Table 1, Figure 2C [i]).4 These observations suggest that Smut0697 is regulated by the competence quorum sensing system, in a manner similar to its homologue in S. pneumoniae.33 The first of the two other proteins with a cin-box in its promoter region was another hypothetical protein, Smut0760, which was up-regulated on average 6.3-fold in biofilm grown cells (Table 1, Figure 3). As with the hypothetical protein, Smut0697, the role of Smut0760 remains unknown. The second protein, SsuRB, that was up-regulated on average 6.8-fold (Table 1, Figure 2C[ii]), forms part of a putative restriction modification system which also contains two methyltransferases, DpnM and Dpn. All three proteins are encoded by an operon homologous to the dpnII operon of S. pneumoniae.44,45 In S. pneumoniae, two transcription products are transcribed from the three genes, dpnM, dpnA and dpnB in the dpnII operon.46 The first transcript is directed from a promoter at the start of the dpnM gene and includes all three genes. The second transcript is initiated from a cin-box promoter located a few bases within the dpnA gene resulting in the competence-pheromone-induced co-transcription of dpnA and dpnB.47 This transcript was not observed in the microarray study of Peterson et al.33 due to the absence of the dpnII operon in the TIGR strain of S. pneumoniae. In S. mutans, an identical cin-box promoter is present in the second gene, dpn, upstream of ssuRB (Figure 3). In S. pneumoniae, DpnM and DpnA methylate the adenine of the double stranded DNA and single stranded DNA sequence 5′-GATC, respectively, while the restriction endonuclease, DpnB, that is homologous to SsuRB in S. mutans, specifically cleaves unmethylated double stranded DNA.47-49 It has been suggested that the primary function of DpnA is to methylate single stranded plasmid DNA taken up by the transformation mechanism in order to provide protection from the DpnB endonuclease when the single stranded DNA is reconstituted into double stranded plasmid DNA in the cytoplasm.47 An alternative hypothesis is that the induction of dpnA during competence facilitates chromosomal exchange of genetic cassettes, since it has been argued that methylation would protect the DNA during chromosomal insertion.50 DNA derived from the lysis of bacteria in close proximity in the biofilm may, however, be utilized in several ways other than for transformation. For instance, exogenous DNA may have a structural role in biofilm formation by binding and stabilizing biofilm cells.51,52 DNA may also be used to repair damaged chromosomal DNA or be metabolized as a source carbon, nitrogen or nucleotides for DNA and RNA synthesis.53,54 Only the restriction endonuclease, SsuRB, of S. mutans was identified as up-regulated in biofilm grown cells. This was most likely Journal of Proteome Research • Vol. 4, No. 6, 2005 2169
research articles due to the fact that the pI of the methylase, Dpn, is 10.4, which would prevent its resolution on 2D gels. In the absence of any evidence for an increase in the methylase, Dpn, it is therefore not possible to predict whether DNA is taken up primarily as a nutrient source or for genetic exchange. However, the presence of Smf, SsbA, and RecA, that protect single stranded DNA during uptake and transformation, would seem to support the latter conclusion. Notwithstanding this suggestion, it is interesting to note that only 0.03% of S. mutans cells growing in a biofilm are transformed55 and that the nucleotide pool in S. mutans appears to be nongrowth limiting (see below). An ATP-binding component of an ABC transporter involved in DNA translocation during competence, ComYA (Smut1804), was also identified, but only on pH 3-11 range gels due to its pI. However, although it was up-regulated 2.4-fold in biofilm grown cells, this increase was not statistically significant (P
