A fast, easy, accurate method for protein quantitation

Beaudet, Matthew P; Ahnert, Nancy; Dallwig, Jason A; Goodman, Terrie; Thomas, Jerald A

doi:10.1096/fasebj.21.6.a1006-d

Cited by 2 publications

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“…The pellet was collected after the removal of ethanol by drying at 60°C and resuspended in 100 μl PBS. The protein and eDNA content were quantified using Qubit Fluorimeter (Invitrogen, Carlsbad, CA, USA), as described (Bajrami et al, 2009; Beaudet et al, 2007). Glucose concentration as a measure of polysaccharide content was quantified by the Dubois method as described elsewhere (Paraneeiswaran et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans

Shukla

Manobala

Rao

2022

Journal of Applied Microbiology

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Aims:To investigate the molecular basis of biofilm formation in a recombinant lab strain of Deinococcus radiodurans with a plasmid harbouring gfp and kan R that acquired the biofilm-forming ability. Methods and results:Deinococcus radiodurans R1 is known as a nonbiofilm former bacterium and so far there are no reports on its biofilm-producing capabilities.In this study, we investigated the molecular basis of biofilm formation in a recombinant strain of D. radiodurans using classical biofilm assays, confocal laser scanning microscopy and real-time PCR. Biochemical analysis of D. radiodurans biofilm matrix revealed that it consisted predominantly of protein and carbohydrate complexes with a little amount of extracellular DNA (eDNA). Furthermore, studies showed that D. radiodurans biofilm formation was enhanced in the presence of 25 mM Ca 2+ , which enhanced the exopolysaccharide and protein content in the biofilm matrix.Enzymatic treatments with proteinase K, alginate lyase and DNase I indicated the involvement of some proteinaceous components to be critical in the biofilm formation. RT-PCR studies showed that increased expression of a surface layer protein SlpA conferred the biofilm ability to D. radiodurans. Conclusion: Overexpression ofSlpA in D. radiodurans conferred the biofilm formation ability to the bacterium, in which a partial role was also played by the recombinant plasmid pKG. It was also shown that the presence of Ca 2+ in the growth medium enhanced SlpA production, thus improving biofilm stability and biofilm maturation of D. radiodurans. Significance and Impact: This study shows how biofilm formation can be augmented in D. radiodurans. The finding has implications for the development of D. radiodurans biofilm-based biotechnological applications.

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Section: Methodsmentioning

confidence: 99%

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans

Shukla

Manobala

Rao

2022

Journal of Applied Microbiology

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Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

Shukla¹,

Toleti²

2017

Indian Journal of Medical Research

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Background & objectives:Among cell surface proteins, biofilm-associated protein (Bap) promotes biofilm development in Staphylococcus aureus strains. The aim of this study was to investigate proteinase-mediated biofilm dispersion in different isolates of S. aureus.Methods:Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA), before and after the proteinase K treatment.Results:Biofilm assay showed that 2 µg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352), but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain). Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 µg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates.Interpretation & conclusions:Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies against S. aureus biofilms.

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A fast, easy, accurate method for protein quantitation

Cited by 2 publications

References 0 publications

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans

Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

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