Biomedical subjects

Arnold S Bleiweis

Publications and source records attributed to Arnold S Bleiweis.

8 recordsLinked to original sources

PubMed2006

Membrane composition changes and physiological adaptation by Streptococcus mutans signal recognition particle pathway mutants.

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 (Deltaffh, DeltascRNA, and DeltaftsY) 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 beta 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 (Deltaffh 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.

Adaptation, Physiological↗

PubMed2005

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2.

The signal recognition particle (SRP)-translocation pathway is conserved in all three domains of life and delivers membrane and secretory proteins to the cytoplasmic membrane or endoplasmic reticulum. We determined the requirement in the cariogenic oral pathogen Streptocococcus mutans of the three universally conserved elements of the SRP pathway: Ffh/SRP54, scRNA, and FtsY/SRalpha. Previously, we reported that insertional interruption of S. mutans ffh was not lethal, but resulted in acid sensitivity. To test whether S. mutans could survive extensive disruption of the SRP pathway, single and double deletions of genes encoding Ffh, scRNA, and FtsY were generated. Without environmental stressors, all mutant strains were viable, but unlike the wild-type, none could initiate growth at pH 5.0 or in 3.5% NaCl. Survival of challenge with 0.3 mM H(2)O(2) was also diminished without ffh. Members of the YidC/Oxa1/Alb3 family are also ubiquitous, involved in the translocation and assembly of membrane proteins, and have been identified in prokaryotes/mitochondria/chloroplasts. Two genes encoding YidC homologs, YidC1 and YidC2, are present in streptococcal genomes with both expressed in S. mutans. Deletion of YidC1 demonstrated no obvious phenotype. Elimination of YidC2 resulted in a stress-sensitive phenotype similar to SRP pathway mutants. Mutants lacking both YidC2 and SRP components were severely impaired and barely able to grow, even in the absence of environmental stress. Here, we report the dispensability of the cotranslational SRP protein translocation system in a bacterium. In S. mutans, this pathway contributes to protection against rapid environmental challenge and may overlap functionally with YidC2.

Bacterial Proteins↗

PubMed2005

Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis.

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.

Chloroform↗

PubMed2004

An ffh mutant of Streptococcus mutans is viable and able to physiologically adapt to low pH in continuous culture.

Previously, we described in Streptococcus mutans strain NG8 a 5-gene operon (sat) that includes ffh, the bacterial homologue of the eukaryotic signal recognition particle (SRP) protein, SR54. A mutation in ffh resulted in acid sensitivity but not loss of viability. In the present study, chemostat-grown cells of the ffh mutant were shown to possess only 26% and 39% of the parental membrane F-ATPase activity and 55% and 75% of parental glucose-phosphotransferase (PTS) activity when pH-7 and pH-5-grown cells, respectively, were assayed. Two-dimensional-gel electrophoretic analyses revealed significant differences in protein profiles between parent and ffh-mutant strains at both pH 5 and pH 7. It appears that the loss of active SRP (Ffh) function, while not lethal, results in substantial alterations in cellular physiology that includes acid tolerance.

Acclimatization↗

PubMed2004

Contribution of the alanine-rich region of Streptococcus mutans P1 to antigenicity, surface expression, and interaction with the proline-rich repeat domain.

Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli, and (v) in E. coli, P1 is secreted in the absence of SecB.

Alanine↗

PubMed2003

Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization, enzymatic activity, and protein-protein interactions.

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.

Actins↗

PubMed2000

A novel beta-glucoside-specific PTS locus from Streptococcus mutans that is not inhibited by glucose.

A regulon from Streptococcus mutans that plays a role in the utilization of beta-glucosides has been isolated, sequenced and subjected to sequence analysis. This regulon encodes a beta-glucoside-specific Enzyme II (EII) component (bglP) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a phospho-beta-glucosidase (bglA) which is responsible for the breakdown of the phospho-beta-glucosides within the cell. Both the bglP and bglA gene products have significant similarity with proteins that have similar functions from Clostridium longisporum, Listeria monocytogenes, Erwinia chrysanthemi, Escherichia coli, Klebsellia oxytoca and Bacillus subtilis. The potential functions of the BglP and BglA proteins are supported by phenotypic data from both S. mutans and E. coli. A chromosomal deletion in S. mutans spanning the bglP and bglA genes resulted in a strain that was unable to hydrolyse the beta-glucoside aesculin in the presence of glucose. When glucose was removed from the medium, the deletion strain regained the ability to break down aesculin. These data suggest that S. mutans possesses an alternative mechanism from the one described in this report for breaking down beta-glucosides. This second mechanism was repressed by glucose while the regulon described here was not. Complementation studies in E. coli CC118 also suggest a potential role for this regulon in the utilization of other beta-glucosides. When a plasmid containing the 8 kb beta-glucoside-specific regulon was transformed into E. coli CC118, the transformed strain was able to break down the beta-glucoside arbutin.

Chromosome Deletion↗

PubMed1999

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Bacterial Proteins↗