Identification and recombinant expression of a cutinase from Papiliotrema laurentii that hydrolyzes natural and synthetic polyesters.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade nature's polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

Edge-Localized Biodeterioration and Secondary Microplastic Formation by Papiliotrema laurentii Unsaturated Biofilm Cells on Polyurethane Films.

Painted environmental surfaces are prone to microbiological colonization with potential coating deterioration induced by the microorganisms. Accurate mechanistic models of these interactions require an understanding of the heterogeneity in which the deterioration processes proceed. Here, unsaturated biofilms (i.e., at air/solid interfaces) of the yeast Papiliotrema laurentii were prepared on polyether polyurethane (PEUR) and polyester-polyether polyurethane (PEST-PEUR) coatings and incubated for up to 33 days at controlled temperature and humidity with no additional nutrients. Transmission micro-Fourier transform infrared microscopy (µFTIR) confirmed preferential hydrolysis of the ester component by the biofilm. Atomic force microscopy combined with infrared nanospectroscopy (AFM-IR) was used to analyze initial PEST-PEUR coating deterioration processes at the single-cell level, including underlying surfaces that became exposed following cell translocation. The results revealed distinct deterioration features that remained localized within ∼10 µm or less of the edges of individual cells and cell clusters. These features comprised depressions of up to ∼300 nm with locally reduced ester/urethane ratios. They are consistent with a formation process initiated by enzymatic ester hydrolysis followed by erosion from water condensation cycles. Further observations included particle accumulation in the broader biofilm vicinity. AFM-IR spectroscopy indicated these to be secondary microplastics consisting of urethane-rich oligomeric aggregates. Overall, multiple contributing factors have been identified that can facilitate differential deterioration rates across the PEST-PEUR surface. Effects of the imposed nutrient conditions on Papiliotrema laurentii physiology were also apparent, with cells developing the characteristics of starvation response, despite the availability of polyester metabolites as a carbon source. The combined results provide new laboratory insights into field-relevant microbiological polymer deterioration mechanisms and biofilm physiology at polymer coating interfaces.

Draft Genome Sequence of Filamentous Fungus Phialemoniopsis curvata, Isolated from Diesel Fuel.

Phialemoniopsis curvata D216 is a filamentous fungus isolated from contaminated diesel fuel. The genome size is 40.3 Mbp with a G+C content of 54.81%. Its genome encodes enzymes and pathways likely involved in the degradation of and survival in fuel, including lipases, fatty acid transporters, and beta oxidation.

3D Printing of Regenerated Silk Fibroin and Antibody-Containing Microstructures via Multiphoton Lithography.

Regenerated silk fibroin, a biopolymer derived from silkworm cocoons, is a versatile material that has been widely explored for a number of applications (e.g., drug delivery, tissue repair, biocompatible electronics substrates, and optics) due to its attractive biochemical properties and processability. Here, we report on the free-form printing of silk-based, 3D microstructures through multiphoton lithography. Utilizing multiphoton lithography in conjunction with specific photoinitiator chemistry and postprint cross-linking, a number of microarchitectures were achieved including self-supporting fibroin arches. Further, the straightforward production of high fidelity and biofunctional protein architectures was enabled through the printing of aqueous fibroin/immunoglobulin solutions.

Carbon Catabolite Repression and Impranil Polyurethane Degradation in Pseudomonas protegens Strain Pf-5.

Polyester polyurethane (PU) coatings are widely used to help protect underlying structural surfaces but are susceptible to biological degradation. PUs are susceptible to degradation by Pseudomonas species, due in part to the degradative activity of secreted hydrolytic enzymes. Microorganisms often respond to environmental cues by secreting enzymes or secondary metabolites to benefit their survival. This study investigated the impact of exposing several Pseudomonas strains to select carbon sources on the degradation of the colloidal polyester polyurethane Impranil DLN (Impranil). The prototypic Pseudomonas protegens strain Pf-5 exhibited Impranil-degrading activities when grown in sodium citrate but not in glucose-containing medium. Glucose also inhibited the induction of Impranil-degrading activity by citrate-fed Pf-5 in a dose-dependent manner. Biochemical and mutational analyses identified two extracellular lipases present in the Pf-5 culture supernatant (PueA and PueB) that were involved in degradation of Impranil. Deletion of the pueA gene reduced Impranil-clearing activities, while pueB deletion exhibited little effect. Removal of both genes was necessary to stop degradation of the polyurethane. Bioinformatic analysis showed that putative Cbr/Hfq/Crc-mediated regulatory elements were present in the intergenic sequences upstream of both pueA and pueB genes. Our results confirmed that both PueA and PueB extracellular enzymes act in concert to degrade Impranil. Furthermore, our data showed that carbon sources in the growth medium directly affected the levels of Impranil-degrading activity but that carbon source effects varied among Pseudomonas strains. This study uncovered an intricate and complicated regulation of P. protegens PU degradation activity controlled by carbon catabolite repression. IMPORTANCE: Polyurethane (PU) coatings are commonly used to protect metals from corrosion. Microbiologically induced PU degradation might pose a substantial problem for the integrity of these coatings. Microorganisms from diverse genera, including pseudomonads, possess the ability to degrade PUs via various means. This work identified two extracellular lipases, PueA and PueB, secreted by P. protegens strain Pf-5, to be responsible for the degradation of a colloidal polyester PU, Impranil. This study also revealed that the expression of the degradative activity by strain Pf-5 is controlled by glucose carbon catabolite repression. Furthermore, this study showed that the Impranil-degrading activity of many other Pseudomonas strains could be influenced by different carbon sources. This work shed light on the carbon source regulation of PU degradation activity among pseudomonads and identified the polyurethane lipases in P. protegens.

The importance of correcting for variable probe-sample interactions in AFM-IR spectroscopy: AFM-IR of dried bacteria on a polyurethane film.

AFM-IR is a combined atomic force microscopy-infrared spectroscopy method that shows promise for nanoscale chemical characterization of biological-materials interactions. In an effort to apply this method to quantitatively probe mechanisms of microbiologically induced polyurethane degradation, we have investigated monolayer clusters of ∼200 nm thick Pseudomonas protegens Pf-5 bacteria (Pf) on a 300 nm thick polyether-polyurethane (PU) film. Here, the impact of the different biological and polymer mechanical properties on the thermomechanical AFM-IR detection mechanism was first assessed without the additional complication of polymer degradation. AFM-IR spectra of Pf and PU were compared with FTIR and showed good agreement. Local AFM-IR spectra of Pf on PU (Pf-PU) exhibited bands from both constituents, showing that AFM-IR is sensitive to chemical composition both at and below the surface. One distinct difference in local AFM-IR spectra on Pf-PU was an anomalous ∼4× increase in IR peak intensities for the probe in contact with Pf versus PU. This was attributed to differences in probe-sample interactions. In particular, significantly higher cantilever damping was observed for probe contact with PU, with a ∼10× smaller Q factor. AFM-IR chemical mapping at single wavelengths was also affected. We demonstrate ratioing of mapping data for chemical analysis as a simple method to cancel the extreme effects of the variable probe-sample interactions.

Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output.

The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28 µW/cm(2)) compared to Luria-Bertani (LB) (0.07 µW/cm(2)) or a defined growth minimal medium (MM) (0.02 µW/cm(2)). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

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.

The utility of Shewanella japonica for microbial fuel cells.

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.

Impedance spectroscopy as a tool for non-intrusive detection of extracellular mediators in microbial fuel cells.

Endogenously produced, diffusible redox mediators can act as electron shuttles for bacterial respiration. Accordingly, the mediators also serve a critical role in microbial fuel cells (MFCs), as they assist extracellular electron transfer from the bacteria to the anode serving as the intermediate electron sink. Electrochemical impedance spectroscopy (EIS) may be a valuable tool for evaluating the role of mediators in an operating MFC. EIS offers distinct advantages over some conventional analytical methods for the investigation of MFC systems because EIS can elucidate the electrochemical properties of various charge transfer processes in the bio-energetic pathway. Preliminary investigations of Shewanella oneidensis DSP10-based MFCs revealved that even low quantities of extracellular mediators significantly influence the impedance behavior of MFCs. EIS results also suggested that for the model MFC studied, electron transfer from the mediator to the anode may be up to 15 times faster than the electron transfer from bacteria to the mediator. When a simple carbonate membrane separated the anode and cathode chambers, the extracellular mediators were also detected at the cathode, indicating diffusion from the anode under open circuit conditions. The findings demonstrated that EIS can be used as a tool to indicate presence of extracellular redox mediators produced by microorganisms and their participation in extracellular electron shuttling.

The influence of acidity on microbial fuel cells containing Shewanella oneidensis.

Microbial fuel cells (MFCs) traditionally operate at pH values between 6 and 8. However, the effect of pH on the growth and electron transfer abilities of Shewanella oneidensis MR-1 (wild-type) and DSP10 (spontaneous mutant), bacteria commonly used in MFCs, to electrodes has not been examined. Miniature MFCs using bare graphite felt electrodes and nanoporous polycarbonate membranes with MR-1 or DSP10 cultures generated >8W/m(3) and approximately 400muA between pH 6-7. The DSP10 strain significantly outperformed MR-1 at neutral pH but underperformed at pH 5. Higher concentrations of DSP10 were sustained at pH 7 relative to that of MR-1, whereas at pH 5 this trend was reversed indicating that cell count was not solely responsible for the observed differences in current. S. oneidensis MR-1 was determined to be more suitable than DSP10 for MFCs with elevated acidity levels. The concentration of riboflavin in the bacterial cultures was reduced significantly at pH 5 for DSP10, as determined by high performance liquid chromatography (HPLC) of the filter sterilized growth media. In addition, these results suggest that mediator biosynthesis and not solely bacterial concentration plays a significant role in current output from S. oneidensis containing MFCs.

Conversion of 2-(4-carboxyphenyl)-6-nitrobenzothiazole to 4-(6-amino-5-hydroxybenzothiazol-2-yl)benzoic acid by a recombinant E. coli strain.

E. coli C43(DE3)pNbzAHabA, expressing nitroreductase and mutase enzymes, converts 2-(4-carboxyphenyl)-6-nitrobenzothiazole to 4-(6-amino-5-hydroxybenzothiazol-2-yl)benzoic acid.

Continuous synthesis of aminophenols from nitroaromatic compounds by combination of metal and biocatalyst.

The combined action of immobilized hydroxylaminobenzene mutase and zinc in a flow-through system catalyzes the conversion of nitroaromatic compounds to the corresponding ortho-aminophenols, including a novel analog of chloramphenicol.

Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols.

The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.

Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups.

Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H(2)(18)O did not indicate any (18)O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R. eutropha JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was (18)O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.