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 Multiple holins contribute to extracellular DNA release in Pseudomonas aeruginosa biofilms

Microbiology ◽  
2021 ◽  
Author(s):  
Amelia L. Hynen ◽  
James J. Lazenby ◽  
George M. Savva ◽  
Laura C. McCaughey ◽  
Lynne Turnbull ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Species ◽  
Extracellular Polymeric Substances ◽  
Bacterial Population ◽  
Extracellular Dna ◽  
Bacterial Biofilms ◽  
Content Type ◽  
Link Type ◽  
Dna Release ◽  
Biofilm Development

Bacterial biofilms are composed 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 Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

PLoS ONE ◽  
2012 ◽  
Vol 7 (10) ◽  
pp. e46718 ◽  
Author(s):  
Theerthankar Das ◽  
Mike Manefield
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Extracellular Dna ◽  
Dna Release

scholarly journals Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

2009 ◽  
Vol 77 (9) ◽  
pp. 3626-3638 ◽  
Author(s):  
Pascale S. Guiton ◽  
Chia S. Hung ◽  
Kimberly A. Kline ◽  
Robyn Roth ◽  
Andrew L. Kau ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Laser Scanning ◽  
Developmental Stages ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Sortase A ◽  
Hydrodynamic Conditions ◽  
Biofilm Production ◽  
Dna Release ◽  
Biofilm Development

ABSTRACT Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.

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.

The role of extracellular polymeric substances in biofilm community assembly and metabolic functions

2020 ◽  
Author(s):  
Yichao Wu
Keyword(s):  
Microbial Community ◽  
Extracellular Polymeric Substances ◽  
Extracellular Dna ◽  
Transcriptional Analysis ◽  
Synthetic Wastewater ◽  
Soluble Microbial Products ◽  
Exogenous Bacteria ◽  
Real Wastewater ◽  
Microbial Products ◽  
Biofilm Development

<p>Compared with the chemically defined synthetic wastewater (SynWW), real wastewater has been reported to exhibit distinct effects on microbial community development. Whether and how soluble microbial products in real wastewater contribute to different effects of synthetic and real wastewater on the fate of exogenous bacteria remains elusive. In this study, using a model wastewater bacterium <em>Comamonas testosteroni</em>, we first examined the influences of microfiltration filter-sterilized real wastewater (MF-WW) and SynWW on the retention of <em>C. testosteroni</em> in established wastewater flocs during bioaugmentation. In bioreactors fed with MF-WW, augmentation of <em>C. testosteroni</em> to wastewater flocs resulted in a substantially higher abundance of the augmented bacterial cells than those fed with SynWW. To identify the soluble microbial products in MF-WW contributing to the observed differences between bioaugmentation reactors fed with MF-WW and SynWW, we examined the effect of MF-WW and SynWW on the growth, floc formation, and biofilm development of <em>C. testosteroni</em>. When <em>C. testosteroni</em> grew in MF-WW, visible flocs formed within 2 h, which is in contrast to cell growth in SynWW where floc formation was not observed. We further demonstrated that the observed differences were mainly attributed to the high molecular weight fraction of the soluble extracellular polymeric substances (EPS) in MF-WW, in particular, proteins and extracellular DNA. The DLVO analysis suggested that, in the presence of soluble EPS, the bacterial cell surface exhibits an increased hydrophobicity and a diminished energy barrier, leading to irreversible attachment of planktonic cells and floc formation. The RNA-seq based transcriptional analysis revealed that, in the presence of soluble EPS, genes involved in nonessential metabolisms were downregulated while genes coding for Cco (cbb3- type) and Cox (aa3-type) oxidases with different oxygen affinities were upregulated, facilitating bacterial survival in flocs. Taken together, this study reveals the mechanisms underlying the contribution of soluble EPS to the recruitment of exogenous bacteria by microbial aggregates and provides implications to bioaugmentation.</p> <p> </p> <p><strong>References:</strong></p> <ol> <li>Wu, Y., Zaiden, N., Liu, X., Mukherjee, M. and Cao, B., 2020. Responses of Exogenous Bacteria to Soluble Extracellular Polymeric Substances in Wastewater: A Mechanistic Study and Implications on Bioaugmentation. Environmental Science & Technology. In press</li> <li>Wu, Y., Cai, P., Jing, X., Niu, X., Ji, D., Ashry, N.M., Gao, C. and Huang, Q., 2019. Soil biofilm formation enhances microbial community diversity and metabolic activity. Environment international, 132, p.105116.</li> </ol>

scholarly journals Influence of High Intensity Focused Ultrasound on the Microstructure and c-di-GMP Signaling of Pseudomonas aeruginosa Biofilms

2020 ◽  
Vol 11 ◽  
Author(s):  
Lakshmi Deepika Bharatula ◽  
Enrico Marsili ◽  
Scott A. Rice ◽  
James J. Kwan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Intensity ◽  
Focused Ultrasound ◽  
Antimicrobial Agents ◽  
Biological Effects ◽  
Biological Response ◽  
High Intensity Focused Ultrasound ◽  
Bacterial Biofilms ◽  
Pressure Amplitude ◽  
Biofilm Development

Bacterial biofilms are typically more tolerant to antimicrobials compared to bacteria in the planktonic phase and therefore require alternative treatment approaches. Mechanical biofilm disruption from ultrasound may be such an alternative by circumventing rapid biofilm adaptation to antimicrobial agents. Although ultrasound facilitates biofilm dispersal and may enhance the effectiveness of antimicrobial agents, the resulting biological response of bacteria within the biofilms remains poorly understood. To address this question, we investigated the microstructural effects of Pseudomonas aeruginosa biofilms exposed to high intensity focused ultrasound (HIFU) at different acoustic pressures and the subsequent biological response. Confocal microscopy images indicated a clear microstructural response at peak negative pressures equal to or greater than 3.5 MPa. In this pressure amplitude range, HIFU partially reduced the biomass of cells and eroded exopolysaccharides from the biofilm. These pressures also elicited a biological response; we observed an increase in a biomarker for biofilm development (cyclic-di-GMP) proportional to ultrasound induced biofilm removal. Cyclic-di-GMP overproducing mutant strains were also more resilient to disruption from HIFU at these pressures. The biological response was further evidenced by an increase in the relative abundance of cyclic-di-GMP overproducing variants present in the biofilm after exposure to HIFU. Our results, therefore, suggest that both physical and biological effects of ultrasound on bacterial biofilms must be considered in future studies.

Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

2011 ◽  
Vol 13 (7) ◽  
pp. 1705-1717 ◽  
Author(s):  
Liang Yang ◽  
Yifan Hu ◽  
Yang Liu ◽  
Jingdong Zhang ◽  
Jens Ulstrup ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Extracellular Polymeric Substances ◽  
Biofilm Development

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.

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