Laboratory growth of denitrifying water column microbial consortia from deep-sea shipwrecks in the northern Gulf of Mexico.

Background: Shipwrecks serve as a rich source for novel microbial populations that have largely remained undiscovered. Low temperatures, lack of sunlight, and the availability of substrates derived from the shipwreck's hull and cargo may provide an environment in which microbes can develop unique metabolic adaptations.   Methods: To test our hypothesis that shipwrecks could influence the microbial population involved in denitrification when a consortium is grown in the laboratory, we collected samples proximate to two steel shipwrecks in the northern Gulf of Mexico. Then under laboratory conditions, we grew two independent denitrifying microbial consortia. Each consortium was grown by using the BART assay system and analyzed based on growth kinetics, ion chromatography and 16S amplicon sequencing. Results: Both denitrifying consortia were different from each other based on varied growth profiles, rates of nitrate utilization and 16S amplicon sequencing. Conclusions: Our observations conclude that the laboratory grown water column microbial consortia from deep-sea shipwrecks in the Gulf of Mexico are able to undergo aggressive denitrification.

Controlling autonomous underwater floating platforms using bacterial fermentation.

Biogenic gas has a wide range of energy applications from being used as a source for crude bio-oil components to direct ignition for heating. The current study describes the use of biogenic gases from Clostridium acetobutylicum for a new application-renewable ballast regeneration for autonomous underwater devices. Uninterrupted (continuous) and blocked flow (pressurization) experiments were performed to determine the overall biogas composition and total volume generated from a semirigid gelatinous matrix. For stopped flow experiments, C. acetobutylicum generated a maximum pressure of 55 psi over 48 h composed of 60 % hydrogen gas when inoculated in a 5 % agar (w/v) support with 5 % glucose (w/v) in the matrix. Typical pressures over 24 h at 318 K ranged from 10 to 33 psi. These blocked flow experiments show for the first time the use of microbial gas production as a way to repressurize gas cylinders. Continuous flow experiments successfully demonstrated how to deliver biogas to an open ballast control configuration for deployable underwater platforms. This study is a starting point for engineering and microbiology investigations of biogas which will advance the integration of biology within autonomous systems.

Enhancing the fouling resistance of biocidal urethane coatings via surface chemistry modulation.

A group of novel cross-linked polyurethane materials with varying ratios of hydroxyl-terminated macrodiols and tethered quaternary ammonium biocides have been prepared. The resulting materials had a wide range of thermal, mechanical, and surface properties, dictated by the macrodiol composition and biocide concentration. The complex interplay between surface chemistry and biocide concentration was shown to have a profound effect on the fouling resistance of these materials. While the combination of quaternary ammonium salt (QAS) diols with poly(tetramethylene oxide) macrodiols did not result in any enhancement of fouling resistance, addition of biocides to poly(ethylene glycol)-containing urethanes resulted in up to a 90% increase in biocidal activity compared to control materials while reducing the ability for microbes to adhere to the surface by an additional 60%. Materials prepared with polybutadiene macrodiols underwent a thermally induced oxidation, resulting in partial decomposition of the quaternary ammonium salt biocide and joint antimicrobial activity arising from remaining QAS and peroxide compounds.

Coatings capable of germinating and neutralizing Bacillus anthracis endospores.

Endospores are formed by various bacterial families, including Bacillus and Clostridium, in response to environmental stresses as a means to survive conditions inhospitable to vegetative growth. Although metabolically inert, the endospore must interact with its environment to determine an optimal time to return to a vegetative state, a process known as germination. Germination has been shown to occur in response to a variety of chemical stimuli from specific nutrient germinants including amino acids, sugars and nucleosides. This process is known to be mediated primarily by the GerA family of spore-specific receptor proteins which initiates a signal transduction cascade that results in a return of oxidative metabolism in response to germinant receptor interactions. Herein, we report the development of a novel coating system capable of germinating B. anthracis endospores, followed by rapid killing of the vegetative bacteria by a novel incorporated amphiphilic biocide. The most effective formulation tested exhibited an ability to germinate and kill B. anthracis endospores and vegetative bacteria, respectively. The formulation reported resulted in a 90% reduction in as little as 5 min, and a 6 log reduction by 45 min.

Biodegradation of medium chain hydrocarbons by Acinetobacter venetianus 2AW immobilized to hair-based adsorbent mats.

The natural attenuation of hydrocarbons can be hindered by their rapid dispersion in the environment and limited contact with bacteria capable of oxidizing hydrocarbons. A functionalized composite material is described herein, that combines in situ immobilized alkane-degrading bacteria with an adsorbent material that collects hydrocarbon substrates, and facilitates biodegradation by the immobilized bacterial population. Acinetobacter venetianus 2AW was isolated for its ability to utilize hydrophobic n-alkanes (C10-C18) as the sole carbon and energy source. Growth of strain 2AW also resulted in the production of a biosurfactant that aided in the dispersion of complex mixtures of hydrophobic compounds. Effective immobilization of strain 2AW to the surface of Ottimat™ adsorbent hair mats via vapor phase deposition of silica provided a stable and reproducible biocatalyst population that facilitates in situ biodegradation of n-alkanes. Silica-immobilized strain 2AW demonstrated ca. 85% removal of 1% (v/v) tetradecane and hexadecane within 24 h, under continuous flow conditions. The methodology for immobilizing whole bacterial cells at the surface of an adsorbent, for in situ degradation of hydrocarbons, has practical application in the bioremediation of oil in water emulsions. Published 2011 American Institute of Chemical Engineers Biotechnol Prog., 2011.

Development of broad-spectrum antimicrobial latex paint surfaces employing active amphiphilic compounds.

With the increase in antibiotic-resistant microbes, the production of self-decontaminating surfaces has become an area of research that has seen a surge of interest in recent years. Such surfaces, when incorporated into commercial products such as children's toys, medical devices and hospital surfaces could reduce the number of infections caused by pathogenic microorganisms. A number of active components for self-decontaminating surfaces have been investigated, including common antibiotics, metal ions, quaternary ammonium salts (QAS), and antimicrobial peptides (AMP). A recent research focus has been development of a wide range of amphiphilic antimicrobial additives that when combined with modern low volatile organic compound (VOC), water-based paints leads to a surface concentration of the active compounds as the coating cures. Herein we report the development of antimicrobial coatings containing a variety of additives, both QAS and AMP that are active against a broad-spectrum of potentially pathogenic bacteria (1-7 log kill), as well as enveloped viruses (2-7 log kill) and fungi (1-2 log kill). Additionally, these additives were compatible with water-dispersed acrylate coatings (latex paint) which have a broad range of real world applicability, and remained active for multiple challenges and when exposed to various cleaning scenarios in which they might encounter in real world situations.

Synthesis and development of a multifunctional self-decontaminating polyurethane coating.

A unique, durable, nonleaching antimicrobial urethane coating possessing energy-dampening properties is reported. Five novel diol-functionalized quaternary ammonium bromide salts were designed, synthesized, and cross-linked with a commercial polyisocyanate to afford novel multifunctional self-decontaminating coatings. Leaching of the antimicrobial into the environment is eliminated because of the biocidal tether. The effectiveness of these molecules to self-concentrate at the air-polymer interface without addition of other surface modifying additives proved extremely advantageous, and consequently resulted in microphase separation as confirmed by AFM. The coatings were designed to continuously decontaminate against a variety of pathogenic bacteria in addition to affording preliminary dampening properties. Minimum inhibitory concentration studies as well as surface antimicrobial evaluations were conducted using both Gram-positive and Gram-negative bacteria. Additionally, viscoelastic properties, hardness, tack, and surface energy measurements were used to correlate with coating performance.

Development of antimicrobial peptides (AMPs) for use in self-decontaminating coatings.

Antimicrobial peptides (AMPs) are a class of short polypeptides usually associated with the host organism's innate immune system. AMPs have been identified in a wide range of host organisms, including plants, amphibians, fish, and humans. These peptides usually consist of 30-100 amino acids and are most often cationic. In addition to a net positive charge, AMPs often are amphipathic, containing both hydrophobic and hydrophilic domains. This property allows for increased interaction with and insertion into negatively charged cell walls and membranes of microbes. Because of the prevalence of antibiotic resistance among common human pathogens, recent research into AMPs has revolved around the attempt to increase the availability of drugs to which microbes are susceptible. Because the mechanism of kill for AMPs is different from that of most conventional antibiotics, which tend to be very specific in their targets, AMPs are thought to be a very attractive future substitute for traditional antibiotics. The development of novel self-decontaminating surfaces containing two AMPs previously isolated from Chrysophrys major is reported. These AMPs, Chrysophsin-1 and -3, demonstrated 1-4 logs kill of both Gram-positive and Gram-negative bacteria when incorporated into control acrylic coating systems.

Evidence for singlet-oxygen generation and biocidal activity in photoresponsive metallic nitride fullerene-polymer adhesive films.

The adhesive properties, as measured by bulk tack analysis, are found to decrease in blends of isomerically pure Sc3N@I(h)-C80 metallic nitride fullerene (MNF) and polystyrene-block-polyisoprene-block-polystyrene (SIS) copolymer pressure-sensitive adhesive under white light irradiation in air. The reduction of tack is attributed to the in situ generation of 1O2 and subsequent photooxidative cross-linking of the adhesive film. Comparisons are drawn to classical fullerenes C60 and C70 for this process. This work represents the first demonstration of 1O2 generating ability in the general class of MNFs (M3N@C80). Additional support is provided for the sensitizing ability of Sc3N@I(h)-C80 through the successful photooxygenation of 2-methyl-2-butene to its allylic hydroperoxides in benzene-d(6) under irradiation at 420 nm, a process that occurs at a rate comparable to that of C(60). Photooxygenation of 2-methyl-2-butene is found to be influenced by the fullerene sensitizer concentration and O2 flow rate. Molar extinction coefficients are reported for Sc3N@I(h)-C80 at 420 and 536 nm. Evaluation of the potential antimicrobial activity of films prepared in this study stemming from the in situ generation of 1O2 led to an observed 1 log kill for select Gram-positive and Gram-negative bacteria.

Surface self-concentrating amphiphilic quaternary ammonium biocides as coating additives.

A variety of amphiphilic quaternary dimethylammonium compounds bearing n-alkyl and oxyethylene groups have been designed and synthesized as antimicrobial additives for use in self-decontaminating surfaces. The effectiveness of these additives stems from a unique ability to self-concentrate at the air-polymer interface without the incorporation of exotic perfluorinated or polymeric functionalities. X-ray photoelectron spectroscopy analysis reveals surface enrichment as high as 18-fold, providing a 7-log reduction of both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The migration to the surface is a consequence of the hydrophobicity of the additive within the hydrophilic polyurethane resin, over which an unprecedented level of control can be exerted by altering the lengths of the n-alkyl and oxyethylene groups. Thus, for the first time, specific surface and bulk coating concentrations can be achieved as desired using a single class of antimicrobial additives.

The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment.

The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.

UL20 protein functions precede and are required for the UL11 functions of herpes simplex virus type 1 cytoplasmic virion envelopment.

Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20- and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment.