Cerrena unicolor Laccases, Genes Expression and Regulation of Activity.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase's transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

Laccase Properties, Physiological Functions, and Evolution.

Discovered in 1883, laccase is one of the first enzymes ever described. Now, after almost 140 years of research, it seems that this copper-containing protein with a number of unique catalytic properties is widely distributed across all kingdoms of life. Laccase belongs to the superfamily of multicopper oxidases (MCOs)-a group of enzymes comprising many proteins with different substrate specificities and diverse biological functions. The presence of cupredoxin-like domains allows all MCOs to reduce oxygen to water without producing harmful byproducts. This review describes structural characteristics and plausible evolution of laccase in different taxonomic groups. The remarkable catalytic abilities and broad substrate specificity of laccases are described in relation to other copper-containing MCOs. Through an exhaustive analysis of laccase roles in different taxa, we find that this enzyme evolved to serve an important, common, and protective function in living systems.

Comparative transcriptomic analysis of Cerrena unicolor revealed differential expression of genes engaged in degradation of various kinds of wood.

To explore the number of enzymes engaged by Cerrena unicolor FCL139 for wood degradation, the transcriptomes of the fungus growing on birch, ash, maple sawdust and the control liquid medium were analyzed. Among 12,966 gene models predicted for the C. unicolor genome, 10,396 all-unigenes were detected, of which 9567 were found to be expressed in each of the tested growth media. The highest number (107) of unique transcripts was detected during fungus growth in the control liquid medium, while the lowest number (11) - in the fungal culture comprising maple saw dust. Analysis of C. unicolor transcriptomes identified numerous genes whose expression differed substantially between the mycelia growing in control medium and each of the sawdust media used, with the highest number (828) of upregulated transcripts observed during the fungus growth on the ash medium. Among the 294 genes that were potentially engaged in wood degradation, the expression of 59 was significantly (p < .01) changed in the tested conditions. The transcripts of 37 of those genes were at least four times more abundant in the cells grown in all sawdust media when compared to the control medium. Upregulated genes coding for cellulases and, to a lower extent, hemicellulases predominated during fungus growth on sawdust. Transcripts encoding cellulolytic enzymes were the most abundant in mycelia grown on birch and maple while lower number of such transcripts was detected in fungus growing on ash. The expression pattern of lignolytic activities-coding genes was strongly dependent on the type of sawdust applied for fungus growth medium.

Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution.

Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described.

Identification and Characterization of Early Mission Phase Microorganisms Residing on the Mars Science Laboratory and Assessment of Their Potential to Survive Mars-like Conditions.

Planetary protection is governed by the Outer Space Treaty and includes the practice of protecting planetary bodies from contamination by Earth life. Although studies are constantly expanding our knowledge about life in extreme environments, it is still unclear what the probability is for terrestrial organisms to survive and grow on Mars. Having this knowledge is paramount to addressing whether microorganisms transported from Earth could negatively impact future space exploration. The objectives of this study were to identify cultivable microorganisms collected from the surface of the Mars Science Laboratory, to distinguish which of the cultivable microorganisms can utilize energy sources potentially available on Mars, and to determine the survival of the cultivable microorganisms upon exposure to physiological stresses present on the martian surface. Approximately 66% (237) of the 358 microorganisms identified are related to members of the Bacillus genus, although surprisingly, 22% of all isolates belong to non-spore-forming genera. A small number could grow by reduction of potential growth substrates found on Mars, such as perchlorate and sulfate, and many were resistant to desiccation and ultraviolet radiation (UVC). While most isolates either grew in media containing ≥10% NaCl or at 4°C, many grew when multiple physiological stresses were applied. The study yields details about the microorganisms that inhabit the surfaces of spacecraft after microbial reduction measures, information that will help gauge whether microorganisms from Earth pose a forward contamination risk that could impact future planetary protection policy. Key Words: Planetary protection-Spore-Bioburden-MSL-Curiosity-Contamination-Mars. Astrobiology 17, 253-265.

Community proteomics provides functional insight into polyhydroxyalkanoate production by a mixed microbial culture cultivated on fermented dairy manure.

Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures (MMCs). To maximize PHA production, MMCs are enriched for bacteria with a high polymer storage capacity through the application of aerobic dynamic feeding (ADF) in a sequencing batch reactor (SBR), which consequently induces a feast-famine metabolic response. Though the feast-famine response is generally understood empirically at a macro-level, the molecular level is less refined. The objective of this study was to investigate the microbial community composition and proteome profile of an enriched MMC cultivated on fermented dairy manure. The enriched MMC exhibited a feast-famine response and was capable of producing up to 40 % (wt. basis) PHA in a fed-batch reactor. High-throughput 16S rRNA gene sequencing revealed a microbial community dominated by Meganema, a known PHA-producing genus not often observed in high abundance in enrichment SBRs. The application of the proteomic methods two-dimensional electrophoresis and LC-MS/MS revealed PHA synthesis, energy generation, and protein synthesis prominently occurring during the feast phase, corroborating bulk solution variable observations and theoretical expectations. During the famine phase, nutrient transport, acyl-CoA metabolism, additional energy generation, and housekeeping functions were more pronounced, informing previously under-determined MMC functionality under famine conditions. During fed-batch PHA production, acetyl-CoA acetyltransferase and PHA granule-bound phasin proteins were in increased abundance relative to the SBR, supporting the higher PHA content observed. Collectively, the results provide unique microbial community structural and functional insight into feast-famine PHA production from waste feedstocks using MMCs.

Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development.

The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high-quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC-MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices.

Bacillus and other spore-forming genera: variations in responses and mechanisms for survival.

The ubiquity of Bacilli endospores in soils facilitates their easy transfer routes to other environments, including cleanrooms and low-biomass sites required by many industries such as food production and processing. A bacterial endospore is a metabolically dormant form of life that is much more resistant to heat, desiccation, lack of nutrients, exposure to UV and gamma radiation, organic chemicals, and oxidizing agents than is a vegetative cell. For example, the heat tolerance of endospores depends on multiple factors such as sporulation temperature, core dehydration, and the presence of minerals and small, acid-soluble proteins (SASPs) in the core. This review describes our current understanding of the persistence mechanisms related to sporeformers' biochemical properties and discusses in detail spores' heat, radiation, and reactive chemical resistance. In addition, it discusses the impact of contamination with spores on many areas of human activity, spore adhesive properties, and biofilm contribution to resistance.

Physiological levels of glucose induce membrane vesicle secretion and affect the lipid and protein composition of Yersinia pestis cell surfaces.

Yersinia pestis grown with physiologic glucose increased cell autoaggregation and deposition of extracellular material, including membrane vesicles. Membranes were characterized, and glucose had significant effects on protein, lipid, and carbohydrate profiles. These effects were independent of temperature and the biofilm-related locus pgm and were not observed in Yersinia pseudotuberculosis.

Fungal laccase, manganese peroxidase and lignin peroxidase: gene expression and regulation.

Extensive research efforts have been dedicated to characterizing expression of laccases and peroxidases and their regulation in numerous fungal species. Much attention has been brought to these enzymes broad substrate specificity resulting in oxidation of a variety of organic compounds which brings about possibilities of their utilization in biotechnological and environmental applications. Research attempts have resulted in increased production of both laccases and peroxidases by the aid of heterologous and homologous expression. Through analysis of promoter regions, protein expression patterns and culture conditions manipulations it was possible to compare and identify common pathways of these enzymes' production and secretion. Although laccase and peroxidase proteins have been crystallized and thoroughly analyzed, there are still a lot of questions remaining about their evolutionary origin and the physiological functions. This review describes the present understanding of promoter sequences and correlation between the observed regulatory effects on laccase, manganese peroxidase and lignin peroxidase genes transcript levels and the presence of specific response elements.

Protection of Bacillus pumilus spores by catalases.

Bacillus pumilus SAFR-032, isolated at spacecraft assembly facilities of the National Aeronautics and Space Administration Jet Propulsion Laboratory, is difficult to kill by the sterilization method of choice, which uses liquid or vapor hydrogen peroxide. We identified two manganese catalases, YjqC and BPUM_1305, in spore protein extracts of several B. pumilus strains by using PAGE and mass spectrometric analyses. While the BPUM_1305 catalase was present in six of the B. pumilus strains tested, YjqC was not detected in ATCC 7061 and BG-B79. Furthermore, both catalases were localized in the spore coat layer along with laccase and superoxide dismutase. Although the initial catalase activity in ATCC 7061 spores was higher, it was less stable over time than the SAFR-032 enzyme. We propose that synergistic activity of YjqC and BPUM_1305, along with other coat oxidoreductases, contributes to the enhanced resistance of B. pumilus spores to hydrogen peroxide. We observed that the product of the catalase reaction, gaseous oxygen, forms expanding vesicles on the spore surface, affecting the mechanical integrity of the coat layer, resulting in aggregation of the spores. The accumulation of oxygen gas and aggregations may play a crucial role in limiting further exposure of Bacilli spore surfaces to hydrogen peroxide or other toxic chemicals when water is present.

Maximizing microbial degradation of perchlorate using a genetic algorithm: Media optimization.

Microbial communities are under constant influence of physical and chemical components in ecosystems. Shifts in conditions such as pH, temperature or carbon source concentration can translate into shifts in overall ecosystem functioning. These conditions can be manipulated in a laboratory setup using evolutionary computation methods such as genetic algorithms (GAs). In work described here, a GA methodology was successfully applied to define sets of environmental conditions for microbial enrichments and pure cultures to achieve maximum rates of perchlorate degradation. Over the course of 11 generations of optimization using a GA, we saw a statistically significant 16.45 and 16.76-fold increases in average perchlorate degradation rates by Dechlorosoma sp. strain KJ and Dechloromonas sp. strain Miss R, respectively. For two bacterial consortia, Pl6 and Cw3, 5.79 and 5.75-fold increases in average perchlorate degradation were noted. Comparison of zero-order kinetic rate constants for environmental conditions in GA-determined first and last generations of all bacterial cultures additionally showed marked increases.

Sterilization of biological pathogens using supercritical fluid carbon dioxide containing water and hydrogen peroxide.

Novel noninvasive techniques for the removal of biological contaminants to generate clean or sterile materials are in demand by the medical, pharmaceutical and food industries. The sterilization method described here uses supercritical fluid carbon dioxide (SF-CO(2)) containing 3.3% water and 0.1% hydrogen peroxide (v/v/v) to achieve from four to eight log viability reduction of all tested microbial species, including vegetative cells, spores and biofilms. The sterilization method employs moderate pressure and temperature (80 atm, 50°C) and a short (30-minute) treatment time. The procedure kills various opportunistic pathogens that often persist in biofilm structures, fungal spores commonly associated with nosocomial infections, and Bacillus pumilus SAFR-032 endospores that are notoriously hard to eradicate by conventional sterilization techniques.

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase.

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

C5-functionalized DNA, LNA, and α-L-LNA: positional control of polarity-sensitive fluorophores leads to improved SNP-typing.

Single nucleotide polymorphisms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X enable detection of SNPs at non-stringent conditions due to differential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal denaturation and optical spectroscopic characteristics of monomer X are compared to the corresponding locked nucleic acid (LNA) and α-L-LNA monomers Y and Z. ONs modified with monomers Y or Z result in a) larger increases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluorescent duplexes due to markedly larger fluorescence emission quantum yields (Φ(F)=0.44-0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of monomers X-Z adopt anti and syn conformations upon hybridization with matched and mismatched targets, respectively. The polarity-sensitive 1-pyrenecarboxamido fluorophore is, thereby, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and α-L-LNA monomers Y and Z influence the glycosidic torsional angle profile leading to altered positional control and photophysical properties of the C5-fluorophore.

Peptide biomarkers as evidence of perchlorate biodegradation.

Perchlorate is a known health hazard for humans, fish, and other species. Therefore, it is important to assess the response of an ecosystem exposed to perchlorate contamination. The data reported here show that a liquid chromatography-mass spectrometry-based proteomics approach for the detection of perchlorate-reducing enzymes can be used to measure the ability of microorganisms to degrade perchlorate, including determining the current perchlorate degradation status. Signature peptides derived from chlorite dismutase (CD) and perchlorate reductase can be used as biomarkers of perchlorate presence and biodegradation. Four peptides each derived from CD and perchlorate reductase subunit A (PcrA) and seven peptides derived from perchlorate reductase subunit B (PcrB) were identified as signature biomarkers for perchlorate degradation, as these sequences are conserved in the majority of the pure and mixed perchlorate-degrading microbial cultures examined. However, chlorite dismutase signature biomarker peptides from Dechloromonas agitata CKB were found to be different from those in other cultures used and should also be included with selected CD biomarkers. The combination of these peptides derived from the two enzymes represents a promising perchlorate presence/biodegradation biomarker system. The biomarker peptides were detected at perchlorate concentrations as low as 0.1 mM and at different time points both in pure cultures and within perchlorate-reducing environmental enrichment consortia. The peptide biomarkers were also detected in the simultaneous presence of perchlorate and an alternate electron acceptor, nitrate. We believe that this technique can be useful for monitoring bioremediation processes for other anthropogenic environmental contaminants with known metabolic pathways.

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile.

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

Pyrene-functionalized triazole-linked 2'-deoxyuridines-probes for discrimination of single nucleotide polymorphisms (SNPs).

Oligonucleotides modified with pyrene-functionalized triazole-linked 2'-deoxyuridines display remarkable hybridization-induced increases in fluorescence emission and enable efficient fluorescent discrimination of SNPs via G-specific quenching.

A method for assaying perchlorate concentration in microbial cultures using the fluorescent dye resazurin.

Low concentrations (microg/L) of the perchlorate anion, ClO(4)(-), have been measured in surface and ground water supplies in many locations throughout the United States. Perchlorate is known to affect the function of the thyroid gland in mammals and its toxicity primarily results from its inhibition of thyroid hormone output. The major sources of perchlorate contamination in surface and ground waters are defense contractors, military installations, propellant manufacturers and agriculture. The currently accepted method of perchlorate analysis, recommended by the US EPA, is neither fast nor easy to use and requires purchase of an expensive high performance ion chromatograph (IC). The novel method described here uses dye resazurin to measure perchlorate reduction by bacterial cultures and bacterial consortia in a high-throughput, multi-well, culture plate format. The method is based on the observation that perchlorate reduction and the decrease of resazurin fluorescence occur simultaneously in perchlorate degrading cultures. The bioassays were performed in anaerobic serum bottles or 96-well plates with constant shaking, using a minimal ATCC medium with 10 mM acetate as electron donor/carbon source and 200 ppm perchlorate as an electron acceptor. Fluorescence measurements with excitation at 570 nm and emission at 590 nm were taken in 20 min intervals. Changes in perchlorate concentration were confirmed using IC. Based on the experimental data, a simple model showing the correlation between perchlorate concentration in microbial culture and resazurin fluorescence level was proposed. Other dyes including redox indicators, reactive azo dyes and electron shuttle chemicals were also tested for comparison and were found less useful.

Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA.

Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases.