Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat

Abstract

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.

Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance

“…Proteins were focussed on narrowrange IPG strips for a total of 141?5 kVh (100 V for 7 h, followed by 300 V for 6 h, 1000 V for 2 h, 2500 V for 2 h, 3500 V for 2 h and 5000 V for 25 h) and on broad-range IPG strips for a total of 79?8 kVh (100 V for 2 h, followed by 300 V for 2 h, 1000 V for 2 h, 2500 V for 2 h, 3500 V for 12 h and 5000 V for 6 h). Following separation in the second dimension on 10-18 % T gradient SDS-PAGE, the gels were stained with Sypro Ruby for image analysis, before being 'double stained' with Coomassie blue CBB G-250 for spot excision, tryptic digestion and mass spectroscopic analysis (Len et al, 2003). A high level of reproducibility of protein spots on Sypro Ruby-stained 2-DGE gels was observed between samples obtained from the the same growth pH (data not shown).…”

“…Peptide mass mapping (PMM) analysis of proteins was undertaken as previously described, making use of the six contigs of the S. mutans UA159 genome downloaded on October 6, 2001 that were translated in all six reading frames (Len et al, 2003). The original six contigs were used in this manner as some genes identified in these contigs were not present in the final annotated version.…”

“…Tryptic in-gel digestion, concentration and desalting of low-copy-number proteins, and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopic analysis were carried out (as previously described) on each protein spot that was differentially or uniquely expressed at pH 7?0 or pH 5?0, or that formed part of the metabolic pathways of interest (Len et al, 2003). Mass spectroscopic analysis of protein spots from each of the replicate 2-DGE gels, at both growth pHs, was used to confirm the identity of the proteins that had been matched by the z3 software.…”

“…When steady state had been achieved, the bacterial contents of the culture vessel were harvested, washed and lyophilized, before aliquots (10 mg dry wt) of cells were treated with mutanolysin (Len et al, 2003). Proteins that were to be separated on acidic immobilized pH gradient (IPG) strips (pH 4?0-6?7) were extracted as previously described (Len et al, 2003), except that 1 % (w/v) amidosulfobetaine-14 (ASB-14) and 65 mM DTT were added to produce a modified solubilizing solution for 2-DGE.…”

“…Proteins that were to be separated on acidic immobilized pH gradient (IPG) strips (pH 4?0-6?7) were extracted as previously described (Len et al, 2003), except that 1 % (w/v) amidosulfobetaine-14 (ASB-14) and 65 mM DTT were added to produce a modified solubilizing solution for 2-DGE. While the addition of these reagents increased the total number of protein spots that could be readily discerned on 2-DGE gels, their inclusion selectively inhibited the extraction or subsequent separation of a small number of weakly expressed proteins that had been previously visualized and/or identified on 2-DGE gels (Len et al, 2003).…”