Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (⌬ffh, ⌬scRNA, and ⌬ftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription-and translation-related proteins, the ␤ subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (⌬ffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.

Glial cell line-derived neurotrophic factor (GDNF) gene transfer is being developed as a treatment for Parkinson's disease (PD). Due to the potential for side effects, external transgene regulation should enhance this strategy's safety profile. Here, we demonstrate dynamic control during long-term expression of GDNF using a recombinant adeno-associated virus (rAAV)-based bicistronic tetracycline (tet)-off construct. Nigrostriatal GDNF overexpression induces body weight alterations in rodents, enabling longitudinal in vivo tracking of GDNF expression after nigral vector delivery. Regulated GDNF expression was highly sensitive to dietary doxycycline (DOX), displaying undetectable striatal GDNF levels at serum DOX levels below those required for antimicrobial activity. However, in the absence of DOX, striatal GDNF levels exceeded levels required for efficacy in PD models. We also demonstrate the absence of a series of known GDNF-associated side effects when using direct intrastriatal vector delivery. Therefore, this single rAAV vector system meets most of the requirements for an experimental reagent for treatment of PD.

Proteasomes are composed of 20S core particles (CPs) of α- and β-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α1 and α2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α1 Thr147, α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α1, thus, revealing a new type of proteasomal modification. Probing the biological role of α1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α1. The α1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.

The alternatively spliced type III extradomain B (EIIIB) of fibronectin (FN) is expressed only during embryogenesis, wound healing and tumorigenesis. The biological function of this domain is unclear. We describe here the first crystal structure of the interface between alternatively spliced EIIIB and its adjacent FN type III domain 8 (FN B-8). The opened CC' loop of EIIIB, and the rotation and tilt of EIIIB allow good access to the FG loop of FN-8, which is normally hindered by the CC' loop of FN-7. In addition, the AGEGIP sequence of the CC'' loop of EIIIB replaces the NGQQGN sequence of the CC' loop of FN-7. Finally, the CC'' loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilaginous development, wound healing, and tumorigenesis, respectively.

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is because of low abundance, poor solubility, and inherent hydrophobicity. In this study, membrane preparations from the Gram-positive bacterium Streptococcus mutans were isolated from protoplasts and by mechanical grinding. Membrane proteins were extracted using a mixture of trifluroethanol and chloroform, solubilized using highly chaotropic buffer containing ASB-14 and Triton X-100 and subjected to two-dimensional gel electrophoresis.

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