scholarly journals Heterologous Inducible Expression ofEnterococcus faecalis pCF10 Aggregation Substance Asc10 inLactococcus lactis and Streptococcus gordonii Contributes to Cell Hydrophobicity and Adhesion to Fibrin

2000 ◽  
Vol 182 (8) ◽  
pp. 2299-2306 ◽  
Author(s):  
Helmut Hirt ◽  
Stanley L. Erlandsen ◽  
Gary M. Dunny
Keyword(s):  
Cell Surface ◽  
Cell Surface Hydrophobicity ◽  
Regulatory System ◽  
Surface Hydrophobicity ◽  
Bacterial Cells ◽  
Streptococcus Gordonii ◽  
Cell Surfaces ◽  
Gene Encoding ◽  
Aggregation Substance

ABSTRACT Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis andStreptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces ofE. faecalis and L. lactis but not on that ofS. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactisrevealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

scholarly journals Characterization of Monolaurin Resistance in Enterococcus faecalis

2007 ◽  
Vol 73 (17) ◽  
pp. 5507-5515 ◽  
Author(s):  
Muriel Dufour ◽  
Janet M. Manson ◽  
Philip J. Bremer ◽  
Jean-Pierre Dufour ◽  
Gregory M. Cook ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Wall Structure ◽  
Transmission Electron ◽  
Serious Infections ◽  
Autolytic Activity

ABSTRACT There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 μg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

The Use of X-Ray Photoelectron Spectroscopy for the Study of Oral Streptococcal Cell Surfaces

1997 ◽  
Vol 11 (4) ◽  
pp. 388-394 ◽  
Author(s):  
H.C. Van Der Mei ◽  
H.J. Busscher
Keyword(s):  
Cell Surface ◽  
Photoelectron Spectroscopy ◽  
Surface Composition ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Contact Angles ◽  
Microbial Cell ◽  
Cell Surfaces ◽  
Water Contact ◽  
X Ray

Physicochemical and structural properties of microbial cell surfaces play an important role in their adhesion to surfaces and are determined by the chemical composition of the outermost cell surface. Many traditional methods used to determine microbial cell wall composition require fractionation of the organisms and consequently do not yield information about the composition of the outermost cell surface. X-ray photoelectron spectroscopy (XPS) measures the elemental composition of the outermost cell surfaces of micro-organisms. The technique requires freeze-drying of the organisms, but, nevertheless, elemental surface concentration ratios of oral streptococcal cell surfaces with peritrichously arranged surface structures showed good relationships with physicochemical properties measured under physiological conditions, such as zeta potentials. Isoelectric points ap-peared to be governed by the relative abundance of oxygen- and nitrogen-containing groups on the cell surfaces. Also, the intrinsic microbial cell-surface hydrophobicity by water contact angles related to the cell-surface composition as by XPS and was highest for strains with an elevated isoelectric point. Inclusion of elemental surface compositions for tufted streptococcal strains caused deterioration of the relationships found. Interestingly, hierarchical cluster analysis on the basis of the elemental surface compositions revealed that, of 36 different streptococcal strains, only four S. rattus as well as nine S. mitis strains were located in distinct groups, well separated from the other streptococcal strains, which were all more or less mixed in one group.

scholarly journals Cell Wall-Anchored CshA Polypeptide (259 Kilodaltons) in Streptococcus gordonii Forms Surface Fibrils That Confer Hydrophobic and Adhesive Properties

1999 ◽  
Vol 181 (10) ◽  
pp. 3087-3095 ◽  
Author(s):  
Roderick McNab ◽  
Helen Forbes ◽  
Pauline S. Handley ◽  
Diane M. Loach ◽  
Gerald W. Tannock ◽  
...  
Keyword(s):  
Cell Surface ◽  
Insertional Mutagenesis ◽  
Cell Surface Hydrophobicity ◽  
Cell Aggregation ◽  
Surface Hydrophobicity ◽  
Streptococcus Gordonii ◽  
Adhesive Properties ◽  
Specific Antibodies ◽  
Structural And Functional Properties ◽  
Human Fibronectin

ABSTRACT It has been shown previously that inactivation of thecshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 ± 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces ofcshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entirecshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologousEnterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity overE. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression inE. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stainedE. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 ± 9.1 nm long that were absent from controlE. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordoniiadhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral “sanguis-like” streptococci.

scholarly journals Analysis of Functional Domains of theEnterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

2001 ◽  
Vol 183 (19) ◽  
pp. 5659-5667 ◽  
Author(s):  
C. M. Waters ◽  
G. M. Dunny
Keyword(s):  
Cell Surface ◽  
High Efficiency ◽  
Cell Surface Hydrophobicity ◽  
Surface Protein ◽  
Cell Aggregation ◽  
Surface Hydrophobicity ◽  
Host Cells ◽  
Functional Domains ◽  
Aggregation Substance ◽  
C Terminus

ABSTRACT Pheromone-inducible aggregation substance (AS) proteins ofEnterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.

Cell surface and exopolymer characterization of laboratory stabilized activated sludge from a beverage bottling plant

2001 ◽  
Vol 43 (6) ◽  
pp. 175-184 ◽  
Author(s):  
S. M. Boyette ◽  
J. M. Lovett ◽  
W. G. Gaboda ◽  
J. A. Soares
Keyword(s):  
Cell Surface ◽  
Activated Sludge ◽  
Surface Charge ◽  
Electrophoretic Mobility ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Food Sources ◽  
Colloid Titration ◽  
High Concentrations

Fermentor-stabilized activated sludge from an industrial beverage bottling plant was grown on three different food sources: normal plant wastewater, plant wastewater containing high sucrose concentrations, and a synthetic glucose-based feed stock. Surface charge, hydrophobicity, and exopolysaccharide composition were measured on the stabilized bacterial flocs. Cell surface charge was measured by electrophoretic mobility, dye exchange titration, and a standard colloid titration, while cell hydrophobicity was determined using the bacterial adhesion to hydrocarbons (BATH) test. Exopolysaccharide profiles were determined by measuring concentrations of glucose, galactose, mannose, glucuronic, and galacturonic acids in digested exopolymer extractions using HPLC. Changes in the physical surface properties of the bacteria and the chemical composition of the extracted exopolymers were correlated with differences in the three food sources. Cell surface hydrophobicity was similar for cultures grown on different plant wastewaters, while the culture grown on synthetic food produced less floc hydrophobicity. Electrophoretic mobility measurements, charge titrations, and dye exchange titrations showed different total surface charge as well as varying charge availability. Additionally, total surface charge and total exopolysaccharide concentrations appeared less dependent on food source than the food-to-mass ratio. High concentrations of biodegradable food produced dispersed growth and high concentrations of exopolysaccharides that contributed to poor settling.

The role of an extracellular polysaccharide produced by the marine Pseudomonas sp. S9 in cellular detachment during starvation

1989 ◽  
Vol 35 (2) ◽  
pp. 309-312 ◽  
Author(s):  
Michael Wrangstadh ◽  
Patricia L. Conway ◽  
Staffan Kjelleberg
Keyword(s):  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Cell Surface Hydrophobicity ◽  
Hydrophobic Surface ◽  
Extracellular Polysaccharide ◽  
Surface Hydrophobicity ◽  
Bulk Phase ◽  
Bacterial Cells ◽  
Pseudomonas Sp

An exopolysaccharide polymer is produced by the marine Pseudomonas sp. S9 in response to complete energy and nutrient starvation. The presence of this polysaccharide on the cell surface and its subsequent release have been shown to be associated with both adhesion and detachment of the bacterial cells. Detachment from a hydrophobic surface was correlated to the presence of the exopolysaccharide on detached S9 cells. The exopolysaccharide was detected, using immunofluorescence microscopy, on surface-bound cells after only 15 min of exogenous energy and nutrient deprivation. This technique did not reveal any significant amounts of exopolysaccharide on starving bulk phase cells prior to 3 h of starvation. Cells that detached after 5.5 h of starvation had low cell surface hydrophobicity values and increased amounts of cell-bound exopolysaccharide. In contrast, cells that became detached during the first 5.5 h of starvation showed increasing hydrophobicity values during prolonged bulk phase starvation.

Sign in / Sign up

Export Citation Format

Share Document