Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

Donald Bren School of Environmental Science and Management, University of California-Santa Barbara, Bren Hall, Santa Barbara, CA 93106-5131, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 10/2005; 71(9):5404-10. DOI: 10.1128/AEM.71.9.5404-5410.2005
Source: PubMed


The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.


Available from: Patricia A Holden, Jan 07, 2015
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    • "Consequently, extracellular DNA (eDNA) released into the environment could be an important organic matter constituent and nutrient source for microorganisms (Pietramellara et al., 2009). Although eDNA has received attention for its roles in bacterial transformation (Paget and Simonet, 1994; Pietramellara et al., 2006) and biofilm formation (Whitchurch et al., 2002; Steinberger and Holden, 2005; B€ ockelmann et al., 2006), the decomposition dynamics of eDNA are not well understood (Nannipieri et al., 2012). Specifically, mineralization rates of eDNA in soil have not been characterized, and the representation of eDNA to the soil metagenome remains speculative. "
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    ABSTRACT: Microbial necromass is an important source of stabilized organic matter in soil, yet the decomposition dynamics of necromass constituents have not been adequately characterized. This includes DNA, a nutrient-rich molecule that when released into the environment as extracellular DNA (eDNA) can be readily used by soil microorganisms. However, the ecological relevance of eDNA as a nutrient source for soil microorganisms is relatively unknown. To address these deficits, we performed a laboratory experiment wherein soils were amended with 13C-labeled eDNA and clay minerals known to interact with DNA (kaolinite and montmorillonite). The amount of eDNA-carbon remaining in the soil declined exponentially over time. Kaolinite amendment decreased eDNA decomposition rates and, after 30 days, retained a higher fraction of eDNA-carbon (∼70% remaining) than control or montmorillonite soils (∼40% remaining), indicating that clay mineral sorption can stabilize eDNA-derived carbon in soil. Sequencing of bacterial 16S rRNA genes showed that during the incubation the relative abundance of the added eDNA's sequence decreased by 98%, 92% and 99% in the control, montmorillonite, and kaolinite amended soils respectively. These results suggest that the fraction of eDNA-carbon that remained in the soil was incorporated into microbial biomass, firmly bound to soil constituents, or fragmented and no longer amenable to sequencing. In addition, the eDNA amendment affected the composition of the bacterial community. Specifically, the relative abundance of select phyla (Planctomycetes and TM7) and genera (e.g., Arthrobacter and Nocardioides) were elevated in soils that received eDNA, suggesting these groups may be particularly effective at degrading eDNA and using it for growth. Taken together, these results indicate that while eDNA is consumed by bacteria in soil, a fraction of eDNA material is resistant to decomposition, particularly when stabilized by soil minerals, suggesting a substantial amount of recalcitrant eDNA could accumulate over time.
    Soil Biology and Biochemistry 04/2015; 86. DOI:10.1016/j.soilbio.2015.03.020 · 3.93 Impact Factor
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    • "It has been proposed to be a major source for the transfer of genetic information (Weinberg & Stotzky, 1972; Graham & Istock, 1978; Nielsen et al., 2007). It has been reported to play a role in the formation of microbial biofilms (Whitchurch et al., 2002; Steinberger & Holden, 2005), in the protection from pathogen attack in root cap 'slime' (Wen et al., 2009; Hawes et al., 2011) and in extracellular traps (Brinkmann et al., 2004; Goldmann & Medina, 2012). Extracellular DNA has also been considered as a relevant source of nutrients for plants (Paungfoo-Lonhienne et al., 2010) and microbes (Finkel & Kolter, 2001; Palchevskiy & Finkel, 2006; Pinchuk et al., 2008). "
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    ABSTRACT: Self-inhibition of growth has been observed in different organisms, but an underlying common mechanism has not been proposed so far. Recently, extracellular DNA (exDNA) has been reported as species-specific growth inhibitor in plants and proposed as an explanation of negative plant–soil feedback. In this work the effect of exDNA was tested on different species to assess the occurrence of such inhibition in organisms other than plants. Bioassays were performed on six species of different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects. Treatments consisted in the addition to the growth substrate of conspecific and heterologous DNA at different concentration levels. Results showed that treatments with conspecific DNA always produced a concentration dependent growth inhibition, which instead was not observed in the case of heterologous DNA. Reported evidence suggests the generality of the observed phenomenon which opens new perspectives in the context of self-inhibition processes. Moreover, the existence of a general species-specific biological effect of exDNA raises interesting questions on its possible involvement in self-recognition mechanisms. Further investigation at molecular level will be required to unravel the specific functioning of the observed inhibitory effects.
    New Phytologist 03/2015; 206(1). DOI:10.1111/nph.13306 · 7.67 Impact Factor
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    • "In this case, a complimentary RNAsequencing-based metatranscriptome analysis that maintains the same potential depth and breadth of data acquisition as the metagenomic analyses would aid in the interpretation of these results. (3) It is likely that the biofilm metagenomic analyses were confounded by the sequencing of accumulated DNA that originated from dead cells or the biochemically complex extracellular polymeric substance that is known to contain large quantities of extracellular DNA (Steinberger & Holden 2005; Klein 2011) and is required for biofilm formation (Whitchurch et al. 2002) (eg bulk extraction bias). "
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    ABSTRACT: Metagenomic and metaproteomic analyses were utilized to determine the composition and function of complex air-water interface biofilms sampled from the hulls of two US Navy destroyers. Prokaryotic community analyses using PhyloChip-based 16S rDNA profiling revealed two significantly different and taxonomically rich biofilm communities (6,942 taxa) in which the majority of unique taxa were ascribed to members of the Gammaproteobacteria, Alphaproteobacteria and Clostridia. Although metagenomic sequencing indicated that both biofilms were dominated by prokaryotic sequence reads (> 91%) with the majority of the bacterial reads belonging to the Alphaproteobacteria, the Ship-1 metagenome harbored greater organismal and functional diversity and was comparatively enriched for sequences from Cyanobacteria, Bacteroidetes and macroscopic eukaryotes, whereas the Ship-2 metagenome was enriched for sequences from Proteobacteria and microscopic photosynthetic eukaryotes. Qualitative liquid chromatography-tandem mass spectrometry metaproteome analyses identified 678 unique proteins, revealed little overlap in species and protein composition between the ships and contrasted with the metagenomic data in that ~80% of classified and annotated proteins were of eukaryotic origin and dominated by members of the Bacillariophyta, Cnidaria, Chordata and Arthropoda (data deposited to the ProteomeXchange, identifier PXD000961). Within the shared metaproteome, quantitative (18)O and iTRAQ analyses demonstrated a significantly greater abundance of structural proteins from macroscopic eukaryotes on Ship-1 and diatom photosynthesis proteins on Ship-2. Photosynthetic pigment composition and elemental analyses confirmed that both biofilms were dominated by phototrophic processes. These data begin to provide a better understanding of the complex organismal and biomolecular composition of marine biofilms while highlighting caveats in the interpretation of stand-alone environmental '-omics' datasets.
    Biofouling 11/2014; 30(10):1211-23. DOI:10.1080/08927014.2014.977267 · 3.42 Impact Factor
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