Structured Review

BioSpec biofilms
A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of <t>biofilms</t> formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P
Biofilms, supplied by BioSpec, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "The Bacterial Biofilm Matrix as a Platform for Protein Delivery"

Article Title: The Bacterial Biofilm Matrix as a Platform for Protein Delivery

Journal: mBio

doi: 10.1128/mBio.00127-12

A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of biofilms formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P
Figure Legend Snippet: A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of biofilms formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P

Techniques Used: Mutagenesis, Activity Assay, Plasmid Preparation, Expressing, Recombinant

An enzymatically active RbmA–ChiA-2–FLAG fusion protein is retained in the biofilm matrix. (A) Immunofluorescent imaging of the distribution of ChiA-2–FLAG, RbmA–ChiA-2–FLAG, RbmA-FLAG, or RbmA-CtxB in a biofilm formed by wild-type V. cholerae carrying a plasmid encoding each of these proteins. The proteins were visualized with an anti-FLAG antibody or anti-CtxB antibody in the case of RbmA-CtxB. Bacterial DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). As a control, a biofilm formed by wild-type V. cholerae carrying an empty vector was developed with an anti-FLAG antibody (CTL) (bar = 10 µM). (B) A magnified view of the distribution of RbmA–ChiA-2–FLAG in the biofilm (bar = 10 µM). (C and D) Chitinase activity measured in the biofilms (C) and supernatants (D) of wild-type V. cholerae carrying an empty vector (CTL) or a plasmid encoding RbmA-CtxB (RbmA-CtxB), ChiA-2–FLAG (ChiA-2), or RbmA–ChiA-2–FLAG (RbmA–ChiA-2). The chitinase activity in the biofilm of the strain expressing RbmA–ChiA-2–FLAG was significantly different from that in all other biofilms ( P ≤ 0.0003). Similarly, chitinase activity in the supernatants of strains expressing either RbmA–ChiA-2–FLAG or ChiA-2–FLAG was significantly different from that of strains carrying the control vector ( P = 0.007 and P = 0.0215, respectively) or the RbmA-CtxB fusion ( P = 0.0025 or P = 0.0149, respectively). The difference in chitinase activity between the supernatants of the strains expressing RbmA–ChiA-2–FLAG and ChiA-2–FLAG was not statistically significant ( P = 0.07).
Figure Legend Snippet: An enzymatically active RbmA–ChiA-2–FLAG fusion protein is retained in the biofilm matrix. (A) Immunofluorescent imaging of the distribution of ChiA-2–FLAG, RbmA–ChiA-2–FLAG, RbmA-FLAG, or RbmA-CtxB in a biofilm formed by wild-type V. cholerae carrying a plasmid encoding each of these proteins. The proteins were visualized with an anti-FLAG antibody or anti-CtxB antibody in the case of RbmA-CtxB. Bacterial DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). As a control, a biofilm formed by wild-type V. cholerae carrying an empty vector was developed with an anti-FLAG antibody (CTL) (bar = 10 µM). (B) A magnified view of the distribution of RbmA–ChiA-2–FLAG in the biofilm (bar = 10 µM). (C and D) Chitinase activity measured in the biofilms (C) and supernatants (D) of wild-type V. cholerae carrying an empty vector (CTL) or a plasmid encoding RbmA-CtxB (RbmA-CtxB), ChiA-2–FLAG (ChiA-2), or RbmA–ChiA-2–FLAG (RbmA–ChiA-2). The chitinase activity in the biofilm of the strain expressing RbmA–ChiA-2–FLAG was significantly different from that in all other biofilms ( P ≤ 0.0003). Similarly, chitinase activity in the supernatants of strains expressing either RbmA–ChiA-2–FLAG or ChiA-2–FLAG was significantly different from that of strains carrying the control vector ( P = 0.007 and P = 0.0215, respectively) or the RbmA-CtxB fusion ( P = 0.0025 or P = 0.0149, respectively). The difference in chitinase activity between the supernatants of the strains expressing RbmA–ChiA-2–FLAG and ChiA-2–FLAG was not statistically significant ( P = 0.07).

Techniques Used: Imaging, Plasmid Preparation, Staining, CTL Assay, Activity Assay, Expressing

2) Product Images from "Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm "

Article Title: Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm

Journal: Infection and Immunity

doi: 10.1128/IAI.71.8.4759-4766.2003

Change in thickness of biofilms with time, as measured by confocal microscopy. The depth of the biofilm represents the mean of five randomly chosen sites within each biofilm.
Figure Legend Snippet: Change in thickness of biofilms with time, as measured by confocal microscopy. The depth of the biofilm represents the mean of five randomly chosen sites within each biofilm.

Techniques Used: Confocal Microscopy

Confocal scanning laser micrographs of the development of S. gordonii Challis DL1 biofilms over 26 days. The biofilms were generated on glass coverslips in 0.5× TY medium supplemented with 10 mM sucrose and were stained immediately prior to microscopy with acridine orange. Numbers indicate the age of the biofilm in days. Magnification, ×63.
Figure Legend Snippet: Confocal scanning laser micrographs of the development of S. gordonii Challis DL1 biofilms over 26 days. The biofilms were generated on glass coverslips in 0.5× TY medium supplemented with 10 mM sucrose and were stained immediately prior to microscopy with acridine orange. Numbers indicate the age of the biofilm in days. Magnification, ×63.

Techniques Used: Generated, Staining, Microscopy

Change in CFU within S. gordonii Challis DL1 biofilms cultured in 0.5× TY medium supplemented with sucrose, which was replaced at 24-h intervals. Cells were enumerated over 30 days of cultivation. Numbers of CFU for biofilms harvested at each time point are expressed as means of triplicate determinations. Error bars represent the standard errors of the means and are not detectable where the error is smaller than the symbol.
Figure Legend Snippet: Change in CFU within S. gordonii Challis DL1 biofilms cultured in 0.5× TY medium supplemented with sucrose, which was replaced at 24-h intervals. Cells were enumerated over 30 days of cultivation. Numbers of CFU for biofilms harvested at each time point are expressed as means of triplicate determinations. Error bars represent the standard errors of the means and are not detectable where the error is smaller than the symbol.

Techniques Used: Cell Culture

3) Product Images from "Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm "

Article Title: Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm

Journal: Infection and Immunity

doi: 10.1128/IAI.71.8.4759-4766.2003

Change in thickness of biofilms with time, as measured by confocal microscopy. The depth of the biofilm represents the mean of five randomly chosen sites within each biofilm.
Figure Legend Snippet: Change in thickness of biofilms with time, as measured by confocal microscopy. The depth of the biofilm represents the mean of five randomly chosen sites within each biofilm.

Techniques Used: Confocal Microscopy

Confocal scanning laser micrographs of the development of S. gordonii Challis DL1 biofilms over 26 days. The biofilms were generated on glass coverslips in 0.5× TY medium supplemented with 10 mM sucrose and were stained immediately prior to microscopy with acridine orange. Numbers indicate the age of the biofilm in days. Magnification, ×63.
Figure Legend Snippet: Confocal scanning laser micrographs of the development of S. gordonii Challis DL1 biofilms over 26 days. The biofilms were generated on glass coverslips in 0.5× TY medium supplemented with 10 mM sucrose and were stained immediately prior to microscopy with acridine orange. Numbers indicate the age of the biofilm in days. Magnification, ×63.

Techniques Used: Generated, Staining, Microscopy

Change in CFU within S. gordonii Challis DL1 biofilms cultured in 0.5× TY medium supplemented with sucrose, which was replaced at 24-h intervals. Cells were enumerated over 30 days of cultivation. Numbers of CFU for biofilms harvested at each time point are expressed as means of triplicate determinations. Error bars represent the standard errors of the means and are not detectable where the error is smaller than the symbol.
Figure Legend Snippet: Change in CFU within S. gordonii Challis DL1 biofilms cultured in 0.5× TY medium supplemented with sucrose, which was replaced at 24-h intervals. Cells were enumerated over 30 days of cultivation. Numbers of CFU for biofilms harvested at each time point are expressed as means of triplicate determinations. Error bars represent the standard errors of the means and are not detectable where the error is smaller than the symbol.

Techniques Used: Cell Culture

4) Product Images from "Environmental Determinants of Vibrio cholerae Biofilm Development"

Article Title: Environmental Determinants of Vibrio cholerae Biofilm Development

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.9.5079-5088.2003

Architecture of biofilms formed in DSW supplemented with CAA with and without Ca 2+ : transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.
Figure Legend Snippet: Architecture of biofilms formed in DSW supplemented with CAA with and without Ca 2+ : transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

Techniques Used: Cellular Antioxidant Activity Assay, Staining, Mutagenesis

Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in 10 mM NaCl-YT, 10 mM NaCl-CAA, and 10 mM NaCl supplemented with CAA and carbohydrates (CRB). This strain carries a chromosomal fusion of the vpsL promoter to lacZ .
Figure Legend Snippet: Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in 10 mM NaCl-YT, 10 mM NaCl-CAA, and 10 mM NaCl supplemented with CAA and carbohydrates (CRB). This strain carries a chromosomal fusion of the vpsL promoter to lacZ .

Techniques Used: Cellular Antioxidant Activity Assay

Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in commercial artificial seawater (ASW) supplemented with CAA and DSW supplemented with CAA. This strain carries a chromosomal fusion of the vpsL promoter to lacZ .
Figure Legend Snippet: Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in commercial artificial seawater (ASW) supplemented with CAA and DSW supplemented with CAA. This strain carries a chromosomal fusion of the vpsL promoter to lacZ .

Techniques Used: Cellular Antioxidant Activity Assay

Representative wild-type V. cholerae strain MO10 (WT) and ΔvpsL mutant PW328 biofilms formed over 18 h in 10 mM NaCl or commercial artificial seawater (ASW) supplemented with either YT or CAA. Biofilms were visualized by crystal violet staining.
Figure Legend Snippet: Representative wild-type V. cholerae strain MO10 (WT) and ΔvpsL mutant PW328 biofilms formed over 18 h in 10 mM NaCl or commercial artificial seawater (ASW) supplemented with either YT or CAA. Biofilms were visualized by crystal violet staining.

Techniques Used: Mutagenesis, Cellular Antioxidant Activity Assay, Staining

Architecture of V. cholerae biofilms formed in 10 mM NaCl supplemented with monosaccharides and/or CAA: transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms in 10 mM NaCl supplemented with CAA and 10 mM NaCl supplemented with CAA and monosaccharides (CRB). Transverse sections were obtained at the level of the substratum. Bars = 10 μm.
Figure Legend Snippet: Architecture of V. cholerae biofilms formed in 10 mM NaCl supplemented with monosaccharides and/or CAA: transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms in 10 mM NaCl supplemented with CAA and 10 mM NaCl supplemented with CAA and monosaccharides (CRB). Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

Techniques Used: Cellular Antioxidant Activity Assay, Staining, Mutagenesis

Related Articles

DNA Extraction:

Article Title: Novel application of the MSSCP method in biodiversity studies
Article Snippet: .. DNA extraction Total DNA was extracted essentially as described [ ], but with the following modifications: combined partial samples of the biofilm (6 g) were homogenized using glass beads in the presence of the extraction buffer by shaking in a Mini Bead-Beater (Bio-Spec Products, Bartesville, U.S.A) three times for 1 min and then repetitive frozing-thawing (after the addition of 20% SDS). .. The DNA was purified using the NucleoSpin Tissue kit (Macherey- Nagel).

Incubation:

Article Title: Taxonomic and chemical assessment of exceptionally abundant rock mine biofilm
Article Snippet: .. Briefly: biofilm samples were homogenized using glass beads in the presence of the extraction buffer by shaking in a MiniBead-Beater (Bio-Spec Products, Bartlesville, OK, USA), subsequently incubated at 37 °C for 30 min with a mixture of lysozyme (10 mg/ml) and zymolyase (0.05 mg/ml), lysed by addition of 20% SDS and frost-thaw repetitive cycles, after which DNA was extracted with chloroform/isoamyl alcohol (24:1) and isopropanol precipitation. .. The DNA was further purified using the NucleoSpinTissue kit (Macherey-Nagel, Düren, Germany).

other:

Article Title: The Bacterial Biofilm Matrix as a Platform for Protein Delivery
Article Snippet: Remaining biofilms were washed with 0.1 M phosphate-buffered saline (PBS) (pH 7.0) and then disrupted with 1-mm-diameter beads (Biospec).

Article Title: Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm
Article Snippet: To enumerate cells in the biofilm phase, the biofilms were dislodged from the substratum by scraping and were transferred to a 2-ml Bead Beater tube (BioSpec Products, Bartlesville, Okla.) containing 0.5 ml of 1-mm-diameter glass beads (BioSpec Products) in 1 ml of PBS.

Infection:

Article Title: Infiltration of Matrix-Non-producers Weakens the Salmonella Biofilm and Impairs Its Antimicrobial Tolerance and Pathogenicity
Article Snippet: .. The biofilm was removed from the wells of microtitre plates with sterile tweezers, disrupted mildly by glass beads of size 0.5 mm diameter with very low speed in a Mini bead beater (BioSpec), and infected orally to BALB/c mice. .. The infectious dose was nearly 107 cells ml−1 when they were administered individually, whereas around 5 × 106 cells ml−1 of each cell type was infected when co-cultured.

Mouse Assay:

Article Title: Infiltration of Matrix-Non-producers Weakens the Salmonella Biofilm and Impairs Its Antimicrobial Tolerance and Pathogenicity
Article Snippet: .. The biofilm was removed from the wells of microtitre plates with sterile tweezers, disrupted mildly by glass beads of size 0.5 mm diameter with very low speed in a Mini bead beater (BioSpec), and infected orally to BALB/c mice. .. The infectious dose was nearly 107 cells ml−1 when they were administered individually, whereas around 5 × 106 cells ml−1 of each cell type was infected when co-cultured.

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    BioSpec biofilm matrix
    Architecture of <t>biofilms</t> formed in DSW supplemented with CAA with and without Ca 2+ : transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.
    Biofilm Matrix, supplied by BioSpec, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biofilm matrix/product/BioSpec
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biofilm matrix - by Bioz Stars, 2020-05
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    85
    BioSpec biofilm associated cells
    Quantification ( Upper ) and phase-contrast microscopy ( Lower ) of wild-type V. cholerae (MO10), Δ mshA mutant, and Δ wbfF mutant <t>biofilms</t> after incubation in DSW medium (including Casamino acids, CAA) for 24 h and then 15 min and 24 h after replacement of bathing medium with DSW medium lacking Ca 2+ (-Ca). Black bars, wild-type V. cholerae ; gray bars, Δ mshA ; striped bars, Δ wbfF . (Scale bars = 4 μm.)
    Biofilm Associated Cells, supplied by BioSpec, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biofilm associated cells/product/BioSpec
    Average 85 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    biofilm associated cells - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    Image Search Results


    Architecture of biofilms formed in DSW supplemented with CAA with and without Ca 2+ : transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

    Journal: Applied and Environmental Microbiology

    Article Title: Environmental Determinants of Vibrio cholerae Biofilm Development

    doi: 10.1128/AEM.69.9.5079-5088.2003

    Figure Lengend Snippet: Architecture of biofilms formed in DSW supplemented with CAA with and without Ca 2+ : transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms. Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

    Article Snippet: In all experiments, planktonic cells were removed, and biofilm cells were dispersed and separated from the biofilm matrix by bead beating at 5,000 rpm for 10 s with 1/3 volume of 1-mm-diameter borosilicate beads (BioSpec).

    Techniques: Cellular Antioxidant Activity Assay, Staining, Mutagenesis

    Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in 10 mM NaCl-YT, 10 mM NaCl-CAA, and 10 mM NaCl supplemented with CAA and carbohydrates (CRB). This strain carries a chromosomal fusion of the vpsL promoter to lacZ .

    Journal: Applied and Environmental Microbiology

    Article Title: Environmental Determinants of Vibrio cholerae Biofilm Development

    doi: 10.1128/AEM.69.9.5079-5088.2003

    Figure Lengend Snippet: Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in 10 mM NaCl-YT, 10 mM NaCl-CAA, and 10 mM NaCl supplemented with CAA and carbohydrates (CRB). This strain carries a chromosomal fusion of the vpsL promoter to lacZ .

    Article Snippet: In all experiments, planktonic cells were removed, and biofilm cells were dispersed and separated from the biofilm matrix by bead beating at 5,000 rpm for 10 s with 1/3 volume of 1-mm-diameter borosilicate beads (BioSpec).

    Techniques: Cellular Antioxidant Activity Assay

    Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in commercial artificial seawater (ASW) supplemented with CAA and DSW supplemented with CAA. This strain carries a chromosomal fusion of the vpsL promoter to lacZ .

    Journal: Applied and Environmental Microbiology

    Article Title: Environmental Determinants of Vibrio cholerae Biofilm Development

    doi: 10.1128/AEM.69.9.5079-5088.2003

    Figure Lengend Snippet: Normalized β-galactosidase activities of biofilm-associated and planktonic wild-type V. cholerae strain PW357 cells grown in commercial artificial seawater (ASW) supplemented with CAA and DSW supplemented with CAA. This strain carries a chromosomal fusion of the vpsL promoter to lacZ .

    Article Snippet: In all experiments, planktonic cells were removed, and biofilm cells were dispersed and separated from the biofilm matrix by bead beating at 5,000 rpm for 10 s with 1/3 volume of 1-mm-diameter borosilicate beads (BioSpec).

    Techniques: Cellular Antioxidant Activity Assay

    Representative wild-type V. cholerae strain MO10 (WT) and ΔvpsL mutant PW328 biofilms formed over 18 h in 10 mM NaCl or commercial artificial seawater (ASW) supplemented with either YT or CAA. Biofilms were visualized by crystal violet staining.

    Journal: Applied and Environmental Microbiology

    Article Title: Environmental Determinants of Vibrio cholerae Biofilm Development

    doi: 10.1128/AEM.69.9.5079-5088.2003

    Figure Lengend Snippet: Representative wild-type V. cholerae strain MO10 (WT) and ΔvpsL mutant PW328 biofilms formed over 18 h in 10 mM NaCl or commercial artificial seawater (ASW) supplemented with either YT or CAA. Biofilms were visualized by crystal violet staining.

    Article Snippet: In all experiments, planktonic cells were removed, and biofilm cells were dispersed and separated from the biofilm matrix by bead beating at 5,000 rpm for 10 s with 1/3 volume of 1-mm-diameter borosilicate beads (BioSpec).

    Techniques: Mutagenesis, Cellular Antioxidant Activity Assay, Staining

    Architecture of V. cholerae biofilms formed in 10 mM NaCl supplemented with monosaccharides and/or CAA: transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms in 10 mM NaCl supplemented with CAA and 10 mM NaCl supplemented with CAA and monosaccharides (CRB). Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

    Journal: Applied and Environmental Microbiology

    Article Title: Environmental Determinants of Vibrio cholerae Biofilm Development

    doi: 10.1128/AEM.69.9.5079-5088.2003

    Figure Lengend Snippet: Architecture of V. cholerae biofilms formed in 10 mM NaCl supplemented with monosaccharides and/or CAA: transverse and vertical cross sections through DAPI-stained wild-type V. cholerae strain MO10 and Δ vpsL mutant PW328 biofilms in 10 mM NaCl supplemented with CAA and 10 mM NaCl supplemented with CAA and monosaccharides (CRB). Transverse sections were obtained at the level of the substratum. Bars = 10 μm.

    Article Snippet: In all experiments, planktonic cells were removed, and biofilm cells were dispersed and separated from the biofilm matrix by bead beating at 5,000 rpm for 10 s with 1/3 volume of 1-mm-diameter borosilicate beads (BioSpec).

    Techniques: Cellular Antioxidant Activity Assay, Staining, Mutagenesis

    Quantification ( Upper ) and phase-contrast microscopy ( Lower ) of wild-type V. cholerae (MO10), Δ mshA mutant, and Δ wbfF mutant biofilms after incubation in DSW medium (including Casamino acids, CAA) for 24 h and then 15 min and 24 h after replacement of bathing medium with DSW medium lacking Ca 2+ (-Ca). Black bars, wild-type V. cholerae ; gray bars, Δ mshA ; striped bars, Δ wbfF . (Scale bars = 4 μm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The Vibrio cholerae O139 O-antigen polysaccharide is essential for Ca2+-dependent biofilm development in sea water

    doi: 10.1073/pnas.2334614100

    Figure Lengend Snippet: Quantification ( Upper ) and phase-contrast microscopy ( Lower ) of wild-type V. cholerae (MO10), Δ mshA mutant, and Δ wbfF mutant biofilms after incubation in DSW medium (including Casamino acids, CAA) for 24 h and then 15 min and 24 h after replacement of bathing medium with DSW medium lacking Ca 2+ (-Ca). Black bars, wild-type V. cholerae ; gray bars, Δ mshA ; striped bars, Δ wbfF . (Scale bars = 4 μm.)

    Article Snippet: Biofilm-associated cells were dispersed by mixing in a Vortex apparatus for 10 sec in the presence of borosilicate glass beads (Biospec).

    Techniques: Microscopy, Mutagenesis, Incubation, Cellular Antioxidant Activity Assay

    Biofilm detachment after medium change from DSW medium to DSW medium lacking Ca 2+ . A quantification of surface association by V. cholerae O139 (MO10), MO10-T4 (T4), V. cholerae O1 El Tor (N16961), V. cholerae O37, V. cholerae O39, V. cholerae O103, V. cholerae O141, V. alginolyticus ( V. a ), V. fluvialis ( V. f ), V. parahaemolyticus ( V. p ), and V. vulnificus ( V. v ) after incubation for 24 h in DSW medium (gray bars) and 5 h after replacement of the bathing medium with DSW medium lacking Ca 2+ (black bars). V. cholerae strain data are labeled with the respective V. cholerae O-antigen designations.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The Vibrio cholerae O139 O-antigen polysaccharide is essential for Ca2+-dependent biofilm development in sea water

    doi: 10.1073/pnas.2334614100

    Figure Lengend Snippet: Biofilm detachment after medium change from DSW medium to DSW medium lacking Ca 2+ . A quantification of surface association by V. cholerae O139 (MO10), MO10-T4 (T4), V. cholerae O1 El Tor (N16961), V. cholerae O37, V. cholerae O39, V. cholerae O103, V. cholerae O141, V. alginolyticus ( V. a ), V. fluvialis ( V. f ), V. parahaemolyticus ( V. p ), and V. vulnificus ( V. v ) after incubation for 24 h in DSW medium (gray bars) and 5 h after replacement of the bathing medium with DSW medium lacking Ca 2+ (black bars). V. cholerae strain data are labeled with the respective V. cholerae O-antigen designations.

    Article Snippet: Biofilm-associated cells were dispersed by mixing in a Vortex apparatus for 10 sec in the presence of borosilicate glass beads (Biospec).

    Techniques: Incubation, Labeling

    Change in thickness of biofilms with time, as measured by confocal microscopy. The depth of the biofilm represents the mean of five randomly chosen sites within each biofilm.

    Journal: Infection and Immunity

    Article Title: Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm

    doi: 10.1128/IAI.71.8.4759-4766.2003

    Figure Lengend Snippet: Change in thickness of biofilms with time, as measured by confocal microscopy. The depth of the biofilm represents the mean of five randomly chosen sites within each biofilm.

    Article Snippet: Cells from the biofilms were disaggregated by scraping the biofilm into a 2-ml microcentrifuge tube (Biospec Products) containing 0.5 ml of 1-mm-diameter glass beads and 1.5 ml of PBS.

    Techniques: Confocal Microscopy

    Confocal scanning laser micrographs of the development of S. gordonii Challis DL1 biofilms over 26 days. The biofilms were generated on glass coverslips in 0.5× TY medium supplemented with 10 mM sucrose and were stained immediately prior to microscopy with acridine orange. Numbers indicate the age of the biofilm in days. Magnification, ×63.

    Journal: Infection and Immunity

    Article Title: Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm

    doi: 10.1128/IAI.71.8.4759-4766.2003

    Figure Lengend Snippet: Confocal scanning laser micrographs of the development of S. gordonii Challis DL1 biofilms over 26 days. The biofilms were generated on glass coverslips in 0.5× TY medium supplemented with 10 mM sucrose and were stained immediately prior to microscopy with acridine orange. Numbers indicate the age of the biofilm in days. Magnification, ×63.

    Article Snippet: Cells from the biofilms were disaggregated by scraping the biofilm into a 2-ml microcentrifuge tube (Biospec Products) containing 0.5 ml of 1-mm-diameter glass beads and 1.5 ml of PBS.

    Techniques: Generated, Staining, Microscopy

    Change in CFU within S. gordonii Challis DL1 biofilms cultured in 0.5× TY medium supplemented with sucrose, which was replaced at 24-h intervals. Cells were enumerated over 30 days of cultivation. Numbers of CFU for biofilms harvested at each time point are expressed as means of triplicate determinations. Error bars represent the standard errors of the means and are not detectable where the error is smaller than the symbol.

    Journal: Infection and Immunity

    Article Title: Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm

    doi: 10.1128/IAI.71.8.4759-4766.2003

    Figure Lengend Snippet: Change in CFU within S. gordonii Challis DL1 biofilms cultured in 0.5× TY medium supplemented with sucrose, which was replaced at 24-h intervals. Cells were enumerated over 30 days of cultivation. Numbers of CFU for biofilms harvested at each time point are expressed as means of triplicate determinations. Error bars represent the standard errors of the means and are not detectable where the error is smaller than the symbol.

    Article Snippet: Cells from the biofilms were disaggregated by scraping the biofilm into a 2-ml microcentrifuge tube (Biospec Products) containing 0.5 ml of 1-mm-diameter glass beads and 1.5 ml of PBS.

    Techniques: Cell Culture

    A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of biofilms formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P

    Journal: mBio

    Article Title: The Bacterial Biofilm Matrix as a Platform for Protein Delivery

    doi: 10.1128/mBio.00127-12

    Figure Lengend Snippet: A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of biofilms formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P

    Article Snippet: Remaining biofilms were washed with 0.1 M phosphate-buffered saline (PBS) (pH 7.0) and then disrupted with 1-mm-diameter beads (Biospec).

    Techniques: Mutagenesis, Activity Assay, Plasmid Preparation, Expressing, Recombinant

    An enzymatically active RbmA–ChiA-2–FLAG fusion protein is retained in the biofilm matrix. (A) Immunofluorescent imaging of the distribution of ChiA-2–FLAG, RbmA–ChiA-2–FLAG, RbmA-FLAG, or RbmA-CtxB in a biofilm formed by wild-type V. cholerae carrying a plasmid encoding each of these proteins. The proteins were visualized with an anti-FLAG antibody or anti-CtxB antibody in the case of RbmA-CtxB. Bacterial DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). As a control, a biofilm formed by wild-type V. cholerae carrying an empty vector was developed with an anti-FLAG antibody (CTL) (bar = 10 µM). (B) A magnified view of the distribution of RbmA–ChiA-2–FLAG in the biofilm (bar = 10 µM). (C and D) Chitinase activity measured in the biofilms (C) and supernatants (D) of wild-type V. cholerae carrying an empty vector (CTL) or a plasmid encoding RbmA-CtxB (RbmA-CtxB), ChiA-2–FLAG (ChiA-2), or RbmA–ChiA-2–FLAG (RbmA–ChiA-2). The chitinase activity in the biofilm of the strain expressing RbmA–ChiA-2–FLAG was significantly different from that in all other biofilms ( P ≤ 0.0003). Similarly, chitinase activity in the supernatants of strains expressing either RbmA–ChiA-2–FLAG or ChiA-2–FLAG was significantly different from that of strains carrying the control vector ( P = 0.007 and P = 0.0215, respectively) or the RbmA-CtxB fusion ( P = 0.0025 or P = 0.0149, respectively). The difference in chitinase activity between the supernatants of the strains expressing RbmA–ChiA-2–FLAG and ChiA-2–FLAG was not statistically significant ( P = 0.07).

    Journal: mBio

    Article Title: The Bacterial Biofilm Matrix as a Platform for Protein Delivery

    doi: 10.1128/mBio.00127-12

    Figure Lengend Snippet: An enzymatically active RbmA–ChiA-2–FLAG fusion protein is retained in the biofilm matrix. (A) Immunofluorescent imaging of the distribution of ChiA-2–FLAG, RbmA–ChiA-2–FLAG, RbmA-FLAG, or RbmA-CtxB in a biofilm formed by wild-type V. cholerae carrying a plasmid encoding each of these proteins. The proteins were visualized with an anti-FLAG antibody or anti-CtxB antibody in the case of RbmA-CtxB. Bacterial DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). As a control, a biofilm formed by wild-type V. cholerae carrying an empty vector was developed with an anti-FLAG antibody (CTL) (bar = 10 µM). (B) A magnified view of the distribution of RbmA–ChiA-2–FLAG in the biofilm (bar = 10 µM). (C and D) Chitinase activity measured in the biofilms (C) and supernatants (D) of wild-type V. cholerae carrying an empty vector (CTL) or a plasmid encoding RbmA-CtxB (RbmA-CtxB), ChiA-2–FLAG (ChiA-2), or RbmA–ChiA-2–FLAG (RbmA–ChiA-2). The chitinase activity in the biofilm of the strain expressing RbmA–ChiA-2–FLAG was significantly different from that in all other biofilms ( P ≤ 0.0003). Similarly, chitinase activity in the supernatants of strains expressing either RbmA–ChiA-2–FLAG or ChiA-2–FLAG was significantly different from that of strains carrying the control vector ( P = 0.007 and P = 0.0215, respectively) or the RbmA-CtxB fusion ( P = 0.0025 or P = 0.0149, respectively). The difference in chitinase activity between the supernatants of the strains expressing RbmA–ChiA-2–FLAG and ChiA-2–FLAG was not statistically significant ( P = 0.07).

    Article Snippet: Remaining biofilms were washed with 0.1 M phosphate-buffered saline (PBS) (pH 7.0) and then disrupted with 1-mm-diameter beads (Biospec).

    Techniques: Imaging, Plasmid Preparation, Staining, CTL Assay, Activity Assay, Expressing