scholarly journals Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix

2009 ◽  
Vol 75 (16) ◽  
pp. 5390-5395 ◽  
Author(s):  
Jianfeng Wu ◽  
Chuanwu Xi
Keyword(s):  
Chemical Treatment ◽  
Genomic Dna ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Enzymatic Treatment ◽  
Extracellular Dna ◽  
Extracellular Matrices ◽  
Treatment Methods ◽  
Biofilm Development ◽  
Biofilm Matrix

ABSTRACT The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.

Extracellular DNA and Outer Membrane Vesicles contribute to P. fluorescens SBW25 air-liquid interface biofilms.

2020 ◽  
Author(s):  
Olena Moshynets ◽  
Airat Kayumov ◽  
Olga Iungin ◽  
Svitlana Rymar ◽  
Ianina Pokholenko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Exogenous Dna ◽  
Rhizosphere Bacterium ◽  
Cellulose Matrix ◽  
Dnase Treatment ◽  
Biofilm Development ◽  
Biofilm Matrix

<p>Outer membrane vesicles (OMVs) and extracellular DNA (eDNA) are important for biofilm formation for many bacteria. OMVs are a perfect transport system to deliver biofilm-related components including eDNA beyond the boundaries of cells, and eDNA itself is an important structural component of biofilms as well as enabling horizontal gene transfer and local adaptation. Both OMVs and eDNA are found in the biofilms produced by the opportunistic human pathogen P. aeruginosa and the plant pathogen P. syringae, but as yet, they have not been reported in the cellulose matrix-based biofilms produced by the related model rhizosphere bacterium Pseudomonas fluorescens SBW25.</p> <p>In this work we have gone back to re-assess the complexity of SBW25 biofilms by looking for evidence of OMVs and eDNA associated with biofilm–formation. OMVs were first imaged by SEM and LC-MC analysis used to identify 51 biofilm matrix-associated proteins of which 12 were also identified in biofilm OMVs. Interestingly, only 5 proteins were identified in both biofilm matrix and OMV samples, but not in planktonic OMVs, suggesting that these may be biofilm-specific components.  </p> <p>We also observed eDNA by CLSM in both the weak and poorly-attached Viscous Mass (VM) and robust and well-attached Wrinkly Spreader (WS) air-liquid (A-L) interface biofilms produced by wild-type SBW25 and the Wrinkly Spreader mutant. The eDNA fraction could be precipitated from biofilm cell-free supernatant samples which demonstrated that WS biofilms had two-fold–higher levels than VM biofilms. DNAse treatment effected the development of both types of biofilm and reduced the strength and attachment levels when added to mature VM and WS biofilms. Testing with exogenous DNA suggests that high molecular weight (HMW) DNA is involved in both strength and attachment, perhaps by surface conditioning and interactions with the primary cellulose matrix common to both biofilms. HMW eDNA could be isolated directly from biofilm supernatants whereas two different HMW size fractions could be isolated from OMVs, presumably, from the outer OMV surface because DNAse treatment led to a substantially reduced DNA signal. This suggest that eDNA persistence and degradation in SBW25 biofilms is complex and eDNA fractions may play different roles in biofilm development, protection and adaptation.</p>

scholarly journals Multiple Holins Contribute to Extracellular DNA Release in Pseudomonas aeruginosa Biofilms

2020 ◽  
Author(s):  
Amelia L. Hynen ◽  
James J. Lazenby ◽  
George M. Savva ◽  
Laura C. McCaughey ◽  
Lynne Turnbull ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sustained Release ◽  
Extracellular Polymeric Substances ◽  
Bacterial Population ◽  
Cell Lysis ◽  
Extracellular Dna ◽  
Cell Cluster ◽  
Bacterial Biofilms ◽  
Dna Release ◽  
Biofilm Development

AbstractBacterial biofilms are comprised of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

scholarly journals NontypeableHaemophilus influenzaereleases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

2017 ◽  
Vol 114 (32) ◽  
pp. E6632-E6641 ◽  
Author(s):  
Joseph A. Jurcisek ◽  
Kenneth L. Brockman ◽  
Laura A. Novotny ◽  
Steven D. Goodman ◽  
Lauren O. Bakaletz
Keyword(s):  
Structural Integrity ◽  
Membrane Vesicle ◽  
Cell Lysis ◽  
Extracellular Dna ◽  
Chronic Obstructive ◽  
Type Iv Pilus ◽  
Membrane Pore ◽  
Type Iv ◽  
Dna Release ◽  
Biofilm Matrix

Biofilms formed by nontypeableHaemophilus influenzae(NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense

2020 ◽  
Vol 367 (4) ◽  
Author(s):  
Víctor I Viruega-Góngora ◽  
Iris S Acatitla-Jácome ◽  
Sandra R Reyes-Carmona ◽  
Beatriz E Baca ◽  
Alberto Ramírez-Mata
Keyword(s):  
Extracellular Matrix ◽  
Biofilm Formation ◽  
Azospirillum Brasilense ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Polar Flagellum ◽  
Matrix Components ◽  
Spatio Temporal ◽  
Biofilm Development ◽  
Biofilm Matrix

ABSTRACT Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

scholarly journals Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance

Antibiotics ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 3
Author(s):  
Cristina Uruén ◽  
Gema Chopo-Escuin ◽  
Jan Tommassen ◽  
Raúl C. Mainar-Jaime ◽  
Jesús Arenas
Keyword(s):  
Close Contact ◽  
Animal Health ◽  
Deep Understanding ◽  
Growth Conditions ◽  
Multidrug Resistant ◽  
Extracellular Dna ◽  
Resistant Bacteria ◽  
Resistance To Antibiotics ◽  
Global Threat ◽  
Biofilm Development

Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth conditions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer. A deep understanding of the mechanisms by which biofilms cause tolerance/resistance to antibiotics helps to develop novel strategies to fight these infections.

scholarly journals Methods to extract the exopolymeric matrix from biofilms: a comparative study

1999 ◽  
Vol 39 (7) ◽  
pp. 243-250 ◽  
Author(s):  
Joana Azeredo ◽  
Valentina Lazarova ◽  
Rosário Oliveira
Keyword(s):  
Organic Carbon ◽  
Comparative Study ◽  
Mixed Culture ◽  
Pure Culture ◽  
Cell Walls ◽  
Extraction Method ◽  
Extraction Methods ◽  
Suitable Method ◽  
The Matrix ◽  
Biofilm Matrix

To study the composition of a biofilm a previous extraction method is required to separate cells from the matrix. There are several methods reported in the literature; however they are not efficient or promote leakage of intracellular material. In this work several extraction methods were assayed in mixed culture and pure culture biofilms and their efficiency was evaluated by the amount of organic carbon, proteins and intracellular material extracted. The results showed that the extraction with glutaraldehyde 3% (w/v) was the most suitable method, extracting great amounts of organic carbon without promoting cell lysis or permeabilization. Glutaraldehyde is a bifunctional reagent that binds to cell walls avoiding their permeabilization and the biofilm matrix is solubilized in the solution.

scholarly journals Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans

2021 ◽  
Vol 9 (11) ◽  
pp. 2308
Author(s):  
Yusuke Iwabuchi ◽  
Tomoyo Nakamura ◽  
Yasuka Kusumoto ◽  
Ryoma Nakao ◽  
Tsutomu Iwamoto ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Membrane Vesicles ◽  
Extracellular Dna ◽  
Tooth Surface ◽  
Initial Ph ◽  
Effects Of Ph ◽  
Low Levels ◽  
Quantitative Changes ◽  
Biofilm Development

Streptococcus mutans releases membrane vesicles (MVs) and induces MV-dependent biofilm formation. Glucosyltransferases (Gtfs) are bound to MVs and contribute to the adhesion and glucans-dependent biofilm formation of early adherent bacteria on the tooth surface. The biofilm formation of S. mutans may be controlled depending on whether the initial pH tends to be acidic or alkaline. In this study, the characteristics and effects of MVs extracted from various conditions {(initial pH 6.0 and 8.0 media prepared with lactic acid (LA) and acetic acid (AA), and with NaOH (NO), respectively)} on the biofilm formation of S. mutans and early adherent bacteria were investigated. The quantitative changes in glucans between primary pH 6.0 and 8.0 conditions were observed, associated with different activities affecting MV-dependent biofilm formation. The decreased amount of Gtfs on MVs under the initial pH 6.0 conditions strongly guided low levels of MV-dependent biofilm formation. However, in the initial pH 6.0 and 8.0 solutions prepared with AA and NO, the MVs in the biofilm appeared to be formed by the expression of glucans and/or extracellular DNA. These results suggest that the environmental pH conditions established by acid and alkaline factors determine the differences in the local pathogenic activities of biofilm development in the oral cavity.

scholarly journals Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10340
Author(s):  
Pacharaporn Angthong ◽  
Tanaporn Uengwetwanit ◽  
Wirulda Pootakham ◽  
Kanchana Sittikankaew ◽  
Chutima Sonthirod ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Genomic Dna ◽  
Extraction Methods ◽  
Marine Organisms ◽  
High Quality ◽  
Sequencing Platform ◽  
Global Food Security ◽  
Long Read ◽  
Ctab Method ◽  
Global Food

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

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