Isolation and Staining Reveal the Presence of Extracellular DNA in Marine Gel Particles.

Marine gel particles (MGP) are amorphous hydrogel exudates from bacteria and microalgae that are ubiquitous in the oceans, but their biochemical composition and function are poorly understood. While dynamic ecological interactions between marine microorganisms and MGPs may result in the secretion and mixing of bacterial extracellular polymeric substances (EPS) such as nucleic acids, compositional studies currently are limited to the identification of acidic polysaccharides and proteins in transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP). Previous studies targeted MGPs isolated by filtration. We developed a new way of isolating MGPs from seawater in liquid suspension and applied it to identify extracellular DNA (eDNA) in North Sea surface seawater. Seawater was filtered onto polycarbonate (PC) filters with gentle vacuum filtration, and then the filtered particles were gently resuspended in a smaller volume of sterile seawater. The resulting MGPs ranged in size from 0.4 to 100 µm in diameter. eDNA was detected by fluorescent microscopy using YOYO-1 (for eDNA), with Nile red (targeting cell membranes) as a counterstain. TOTO-3 was also used to stain eDNA, with ConA to localise glycoproteins and SYTO-9 for the live/dead staining of cells. Confocal laser scanning microscopy (CLSM) revealed the presence of proteins and polysaccharides. We found eDNA to be universally associated with MGPs. To further elucidate the role of eDNA, we established a model experimental MGP system using bacterial EPS from Pseudoalteromonas atlantica that also contained eDNA. Our results clearly demonstrate the occurrence of eDNA in MGPs, and should aid furthering our understanding of the micro-scale dynamics and fate of MGPs that underly the large-scale processes of carbon cycling and sedimentation in the ocean.

Bacterial colonisation of plastic in the Rockall Trough, North-East Atlantic: An improved understanding of the deep-sea plastisphere.

Plastic pollution has now been found within multiple ecosystems across the globe. Characterisation of microbial assemblages associated with marine plastic, or the so-called 'plastisphere', has focused predominantly on plastic in the epipelagic zone. Whether this community includes taxa that are consistently enriched on plastic compared to surrounding non plastic surfaces is unresolved, as are the ecological implications. The deep sea is likely a final sink for most of the plastic entering the ocean, yet there is limited information on microbial colonisation of plastic at depth. The aim of this study was to investigate deep-sea microbial communities associated with polystyrene (PS) and polyurethane (PU) with Bath stone used as a control. The substrates (n = 15) were deployed in the Rockall Trough (Atlantic), and recovered 420 days later from a depth of 1796 m. To characterise the bacterial communities, 16S rRNA genes were sequenced using the Illumina MiSeq platform. A dominant core microbiome (taxa shared across all substrates) comprised 8% of total ASVs (amplicon sequence variant) and accounted for 92% of the total community reads. This suggests that many commonly reported members of the plastisphere are simply opportunistic which freely colonise any hard surface. Transiently associated species consisted of approximately 7% of the total community. Thirty genera were enriched on plastic (P < 0.05), representing 1% of the total community. The discovery of novel deep-sea enriched taxa included Aurantivirga, Algivirga, IheB3-7, Spirosoma, HTCC5015, Ekhidna and Calorithrix on PS and Candidatus Obscuribacter, Haloferula, Marine Methylotrophic Group 3, Aliivibrio, Tibeticola and Dethiosulfatarculus on PU. This small fraction of the microbiome include taxa with unique metabolic abilities and show how bacterial communities can be shaped by plastic pollution at depth. This study outlines a novel approach in categorising the plastisphere to elucidate the ecological implications of enriched taxa that show an affinity for colonising plastic.

Nonlinear rheological characteristics of single species bacterial biofilms.

Bacterial biofilms in natural and artificial environments perform a wide array of beneficial or detrimental functions and exhibit resistance to physical as well as chemical perturbations. In dynamic environments, where periodic or aperiodic flows over surfaces are involved, biofilms can be subjected to large shear forces. The ability to withstand these forces, which is often attributed to the resilience of the extracellular matrix. This attribute of the extracellular matrix is referred to as viscoelasticity and is a result of self-assembly and cross-linking of multiple polymeric components that are secreted by the microbes. We aim to understand the viscoelastic characteristic of biofilms subjected to large shear forces by performing Large Amplitude Oscillatory Shear (LAOS) experiments on four species of bacterial biofilms: Bacillus subtilis, Comamonas denitrificans, Pseudomonas fluorescens and Pseudomonas aeruginosa. We find that nonlinear viscoelastic measures such as intracycle strain stiffening and intracycle shear thickening for each of the tested species, exhibit subtle or distinct differences in the plot of strain amplitude versus frequency (Pipkin diagram). The biofilms also exhibit variability in the onset of nonlinear behaviour and energy dissipation characteristics, which could be a result of heterogeneity of the extracellular matrix constituents of the different biofilms. The results provide insight into the nonlinear rheological behaviour of biofilms as they are subjected to large strains or strain rates; a situation that is commonly encountered in nature, but rarely investigated.

Aggregation Pheromone for an Invasive Mussel Consists of a Precise Combination of Three Common Purines.

Most marine benthic invertebrates have a pelagic larval phase, after which they settle preferentially on or near conspecific adults, forming aggregations. Although settlement pheromones from conspecific adults have been implicated as critical drivers of aggregation for more than 30 years, surprisingly few have been unambiguously identified. Here we show that in the invasive dreissenid mussel Mytilopsis sallei (an ecological and economic pest), three common purines (adenosine, inosine, and hypoxanthine) released from adults in a synergistic and precise ratio (1:1.125:3.25) serve as an aggregation pheromone by inducing conspecific larval settlement and metamorphosis. Our results demonstrate that simple common metabolites can function as species-specific pheromones when present in precise combinations. This study provides important insights into our understanding of the ecology and communication processes of invasive organisms and indicates that the combination and ratio of purines might be critical for purine-based signaling systems that are fundamental and widespread in nature.

Importance of Water-Volume on the Release of Microplastic Fibers from Laundry.

The influence of laundry washing parameters on the release of microfibers (MF) from polyester textiles was studied. These fibers are an important type of microplastic pollution. However, the factors which affect MF release during laundry are poorly understood and more rigorous methods for quantifying this release are needed. A novel method was therefore developed using a tergotometer with eight 1000 mL washing vessels and the CIELab color space measure of lightness (L*). L* was related to the mass of released MFs by creating a calibration curve to quantify the amounts of MFs released from textiles during washing. This method was used to investigate the effect of water-volume, agitation, temperature, and duration of the wash on MF release. Counterintuitively, increased water-volume, characteristic of European "delicate" cycles, resulted in the greatest release of MFs. Full-scale testing was then carried out using domestic washing machines with real consumer cycles to determine the effect of cycle type on MF release. In the first wash, delicate wash cycles released 800 000 more MFs (94 mg/kg) per wash than a lower water-volume standard wash and also increased MF release in subsequent washing cycles (P < 0.05). These results indicate that a high water-volume-to-fabric ratio is the most influential factor for MF release, rather than agitation as previously thought. Therefore, consumers can reduce MF release by avoiding high water-volume washes (delicate cycles), transitioning to appliances that use a lower water-volume (North American high-efficiency washing machines), and ensuring that full wash loads are used.

Regulating, Measuring, and Modeling the Viscoelasticity of Bacterial Biofilms

Biofilms occur in a broad range of environments under heterogeneous physicochemical conditions, such as in bioremediation plants, on surfaces of biomedical implants, and in the lungs of cystic fibrosis patients. In these scenarios, biofilms are subjected to shear forces, but the mechanical integrity of these aggregates often prevents their disruption or dispersal. Biofilms' physical robustness is the result of the multiple biopolymers secreted by constituent microbial cells which are also responsible for numerous biological functions. A better understanding of the role of these biopolymers and their response to dynamic forces is therefore crucial for understanding the interplay between biofilm structure and function. In this paper, we review experimental techniques in rheology, which help quantify the viscoelasticity of biofilms, and modeling approaches from soft matter physics that can assist our understanding of the rheological properties. We describe how these methods could be combined with synthetic biology approaches to control and investigate the effects of secreted polymers on the physical properties of biofilms. We argue that without an integrated approach of the three disciplines, the links between genetics, composition, and interaction of matrix biopolymers and the viscoelastic properties of biofilms will be much harder to uncover.

Secretion of DNases by Marine Bacteria: A Culture Based and Bioinformatics Approach.

The vast majority of bacteria present in the natural environment are present in the form of aggregates and/or biofilms. Microbial aggregates are ubiquitous in the marine environment and are inhabited by diverse microbial communities which often express intense extracellular enzymatic activities. However, the secretion of an important group of enzymes, DNases, by bacteria from marine aggregates has not been studied, despite the importance of these aggregates in biogeochemical cycling of nutrients in the oceans. In this work, we therefore, employed both culture-based and bioinformatics approaches to understand the diversity of bacterial DNases in marine bacterioplankton. We found that 34% of 345 strains of attached and non-attached marine bacteria showed extracellular DNase activity. Most of these isolates belong to Proteobacteria (53%) and Firmicutes (34%). Secretion of DNases by bacteria isolated from marine gel particles (MGP) is reported here for the first time. Then, to further understand the wider diversity of the potential to produce DNases, sequences were compared using 2316 whole genome and 42 metagenome datasets. Thirty-nine different taxonomic groups corresponding to 10 bacterial phyla were found to encode genes responsible for DNase secretion. This study highlights the unexpected and widespread presence of DNase secretion in bacteria in general and in MGP more specifically. This has important implications for understanding the dynamics and fate of marine microbial aggregates in the oceans.

UV Resistance of bacteria from the Kenyan Marine cyanobacterium Moorea producens.

UV resistance of bacteria isolated from the marine cyanobacterium Moorea producens has not been observed previously, findings which highlight how unsafe germicidal UV irradiation for sterilization of air, food, and water could be. Further, UV resistance of Bacillus licheniformis is being observed for the first time. This study focused on bacteria isolated from the marine cyanobacterium M. producens collected off the Kenyan coast at Shimoni, Wasini, Kilifi, and Mida. UV irradiance of isolates (302 nm, 70 W/m2 , 0-1 hr) established B. licheniformis as the most UV resistant strain, with the following order of taxon resistance: Bacilli> Î³ proteobacteria > Actinobacteria. UV resistance was independent of pigmentation. The maximum likelihood phylogenetic distance determined for both B. licheniformis and Bacillus aerius relative to M. producens CCAP 1446/4 was 2.0. Survival of B. licheniformis upon UV irradiance followed first-order kinetics (k = 0.035/min, R2  = 0.88). Addition of aqueous extracts (2, 10, 20 and 40 mg/ml) of this B. licheniformis strain on the less resistant Marinobacterium stanieri was not significant, however, the commercial sunscreen benzophenone-3 (BP-3) positive control and the time of irradiance were significant. Detection of bacteria on M. producens filaments stained with acridine orange confirmed its nonaxenic nature. Although the chemistry of UV resistance in cyanobacteria has been studied in depth revealing for example the role of mycosporine like amino acids (MAAs) in UV resistance less is known about how bacteria resist UV irradiation. This is of interest since cyanobacteria live in association with bacteria.

Evolutionary conservation of the antimicrobial function of mucus: a first defence against infection.

Mucus layers often provide a unique and multi-functional hydrogel interface between the epithelial cells of organisms and their external environment. Mucus has exceptional properties including elasticity, changeable rheology and an ability to self-repair by re-annealing, and is therefore an ideal medium for trapping and immobilising pathogens and serving as a barrier to microbial infection. The ability to produce a functional surface mucosa was an important evolutionary step, which evolved first in the Cnidaria, which includes corals, and the Ctenophora. This allowed the exclusion of non-commensal microbes and the subsequent development of the mucus-lined digestive cavity seen in higher metazoans. The fundamental architecture of the constituent glycoprotein mucins is also evolutionarily conserved. Although an understanding of the biochemical interactions between bacteria and the mucus layer are important to the goal of developing new antimicrobial strategies, they remain relatively poorly understood. This review summarises the physicochemical properties and evolutionary importance of mucus, which make it so successful in the prevention of bacterial infection. In addition, the strategies developed by bacteria to counteract the mucus layer are also explored.

Crystal structure of NucB, a biofilm-degrading endonuclease.

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ßßα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

Enhanced eicosapentaenoic acid production by a new deep-sea marine bacterium Shewanella electrodiphila MAR441T.

Omega-3 fatty acids are products of secondary metabolism, essential for growth and important for human health. Although there are numerous reports of bacterial production of omega-3 fatty acids, less information is available on the biotechnological production of these compounds from bacteria. The production of eicosapentaenoic acid (EPA, 20:5ω3) by a new species of marine bacteria Shewanella electrodiphila MAR441T was investigated under different fermentation conditions. This strain produced a high percentage (up to 26%) of total fatty acids and high yields (mg / g of biomass) of EPA at or below the optimal growth temperature. At higher growth temperatures these values decreased greatly. The amount of EPA produced was affected by the carbon source, which also influenced fatty acid composition. This strain required Na+ for growth and EPA synthesis and cells harvested at late exponential or early stationary phase had a higher EPA content. Both the highest amounts (20 mg g-1) and highest percent EPA content (18%) occurred with growth on L-proline and (NH4)2SO4. The addition of cerulenin further enhanced EPA production to 30 mg g-1. Chemical mutagenesis using NTG allowed the isolation of mutants with improved levels of EPA content (from 9.7 to 15.8 mg g-1) when grown at 15°C. Thus, the yields of EPA could be substantially enhanced without the need for recombinant DNA technology, often a commercial requirement for food supplement manufacture.

Chitosan-zinc oxide nanocomposite coatings for the prevention of marine biofouling.

Marine biofouling is a worldwide problem affecting maritime industries. Global concerns about the high toxicity of antifouling paints have highlighted the need to develop less toxic antifouling coatings. Chitosan is a natural polymer with antimicrobial, antifungal and antialgal properties that is obtained from partial deacetylation of crustacean waste. In the present study, nanocomposite chitosan-zinc oxide (chitosan-ZnO) nanoparticle hybrid coatings were developed and their antifouling activity was tested. Chitosan-ZnO nanoparticle coatings showed anti-diatom activity against Navicula sp. and antibacterial activity against the marine bacterium Pseudoalteromonas nigrifaciens. Additional antifouling properties of the coatings were investigated in a mesocosm study using tanks containing natural sea water under controlled laboratory conditions. Each week for four weeks, biofilm was removed and analysed by flow cytometry to estimate total bacterial densities on the coated substrates. Chitosan-ZnO hybrid coatings led to better inhibition of bacterial growth in comparison to chitosan coatings alone, as determined by flow cytometry. This study demonstrates the antifouling potential of chitosan-ZnO nanocomposite hybrid coatings, which can be used for the prevention of biofouling.

Shewanella electrodiphila sp. nov., a psychrotolerant bacterium isolated from Mid-Atlantic Ridge deep-sea sediments.

Strains MAR441(T) and MAR445 were isolated from Mid-Atlantic Ridge sediments from a depth of 2734 m, and were found to belong to the genus Shewanella. The strains were rod-shaped, pigmented, non-motile and capable of anaerobic growth either by fermentation of carbohydrates or by anaerobic respiration. The strains utilized a variety of electron acceptors, including nitrate and ferric compounds, and could utilize peptone when grown anaerobically in a two-chambered microbial fuel cell, which used carbon cloth electrodes and delivered a stable power output of ,150-200 mW m(-2). The major fatty acids were typical of the genus Shewanella, with major components C13 : 0, iso-C13 : 0, iso-C15 : 0, C16 : 0, C16 : 1ω7c, C18 : 1ω7c and C20 : 5ω3 fatty acids. The DNA G+C content of strains MAR441(T) and MAR445 was 42.4 mol%. 16S rRNA gene sequence analysis indicated that strains MAR441(T) and MAR445 were most closely related to Shewanella olleyana (sequence similarities 97.9% to the type strain). DNA-DNA hybridization demonstrated only 15.6-37.2% relatedness between strain MAR441(T) and the type strains of related species of the genus Shewanella. Phenotypic characteristics confirmed that these isolates constituted a novel species of the genus Shewanella, for which the name Shewanella electrodiphila sp. nov. is proposed; the type strain is MAR441(T) (5ATCC BAA-2408(T) = DSM 24955(T)).

Extracellular DNA in oral microbial biofilms.

The extracellular matrix of microbial biofilms is critical for surface adhesion and nutrient homeostasis. Evidence is accumulating that extracellular DNA plays a number of important roles in biofilm integrity and formation on hard and soft tissues in the oral cavity. Here, we summarise recent developments in the field and consider the potential of targeting DNA for oral biofilm control.

The first GCC Marine Biotechnology Symposium: Emerging Opportunities and Future Perspectives.

With its diverse, living marine resources and rapidly growing educational and research infrastructure, the Sultanate of Oman is well-positioned to take advantage of the commercial opportunities presented by marine biotechnology. In recognition of potential development, an international symposium, Marine Biotechnology-Emerging Opportunities and Future Perspectives, was held in Muscat, November 12-13, 2013. Three keynote addresses were given, 23 oral presentations made, and a poster exhibition held. The final session reviewed national and regional issues, and the delegates agreed informally on a number of future actions. The potential for future development of marine biotechnology was recognized by all delegates, and following the symposium, they were surveyed for their views on how best to sustain and develop new activities. One hundred percent of respondents found the meeting useful and would support future symposia in the region. Fifty-one percent of Omani respondents recognized major organizational challenges and obstacles to the development of marine biotechnology compared with 23 % of overseas respondents. The need for greater collaboration between research institutions within the GCC region was recognized by 98 % of all respondents. The presentations and survey outcomes are reviewed in this paper.

Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis.

BACKGROUND: The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. METHODS/PRINCIPAL FINDINGS: Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. CONCLUSION/SIGNIFICANCE: Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.

Use of physiological information and process optimisation enhances production of extracellular nuclease by a marine strain of Bacillus licheniformis.

The extracellular nuclease, NucB, from Bacillus licheniformis, can digest extracellular DNA in biofilms, causing biofilm dispersal, and may therefore be used commercially to remove biofilms. However, producing quantities of this secreted peptide is difficult and our aim was therefore to improve its laboratory scale production. This study builds on our understanding of B. licheniformis physiology to enhance NucB production. The addition of manganese, which triggers sporulation and enhances NucB expression, lead to a 5-fold increase in NucB production. Optimisation via Placket-Burman design of experiments identified 3 significant medium components and a subsequent Central Composite Design, to determine the optimum levels of these components, resulted in a 10-fold increase to 471U/ml. The optimal phosphate concentration was less than 0.3mM as this is known to inhibit nuclease production. The use of physiologically relevant information combined with optimisation represents a promising approach to increased enzyme production, which may also be widely applicable.

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry.

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.

Enhanced electricity production by use of reconstituted artificial consortia of estuarine bacteria grown as biofilms.

Microbial fuel cells (MFCs) can convert organic compounds directly into electricity by catalytic oxidation, and although MFCs have attracted considerable interest, there is little information on the electricity-generating potential of artificial bacterial biofilms. We have used acetate-fed MFCs inoculated with sediment, with two-chamber bottles and carbon cloth electrodes to deliver a maximum power output of ~175 mW · m(-2) and a stable power output of ~105 mW · m(-2). Power production was by direct transfer of electrons to the anode from bacterial consortia growing on the anode, as confirmed by cyclic voltammetry (CV) and scanning electron microscopy (SEM). Twenty different species (74 strains) of bacteria were isolated from the consortium under anaerobic conditions and cultured in the laboratory, of which 34% were found to be exoelectrogens in single-species studies. Exoelectrogenesis by members of the genera Vibrio , Enterobacter , and Citrobacter and by Bacillus stratosphericus was confirmed, by use of culture-based methods, for the first time. An MFC with a natural bacterial consortium showed higher power densities than those obtained with single strains. In addition, the maximum power output could be further increased to ~200 mW · m(-2) when an artificial consortium consisting of the best 25 exoelectrogenic isolates was used, demonstrating the potential for increased performance and underlying the importance of artificial biofilms for increasing power output.