How to Deal with PCR Contamination in Molecular Microbial Ecology

Microbial ecology studies often use broad-range PCR primers to obtain community profiles. Contaminant microbial DNA present in PCR reagents may therefore be amplified together with template DNA, resulting in unrepeatable data which may be difficult to interpret, especially when template DNA is present at low levels. One possible decontamination method consists in pre-treating PCR mixes with restriction enzymes before heat-inactivating those enzymes prior to the start of the PCR. However, this method has given contrasting results, including a reduction in PCR sensitivity. In this study, we tested the efficiency of two different enzymes (DNase 1 and Sau3AI) as well as the effect of dithiothreitol (DTT), a strong reducing agent, in the decontamination procedure. Our results indicate that enzymatic treatment does reduce contamination levels. However, DNase 1 caused substantial reductions in the bacterial richness found in communities, which we interpret as a result of its incomplete inactivation by heat treatment. DTT did help maintain bacterial richness in mixes treated with DNase 1. No such issues arose when using Sau3AI, which therefore seems a more appropriate enzyme. In our study, four operational taxonomic units (OTU) decreased in frequency and relative abundance after treatment with Sau3AI and hence are likely to represent contaminant bacterial DNA. We found higher within-sample similarity in community structure after treatment with Sau3AI, probably better reflecting the initial bacterial communities. We argue that the presence of contaminant bacterial DNA may have consequences in the interpretation of ecological data, especially when using low levels of template DNA from highly diverse communities. We advocate the use of such decontaminating approaches as a standard procedure in microbial ecology.     

Isolated faecal bacterial communities found for Weddell seals,Leptonychotes weddellii,at White Island,McMurdo Sound,Antarctica

The microbial diversity of faecal communities co-existing with mega fauna is not well understood even though these faecal communities are critical for health and development. Additionally, the transfer of microbial taxa among host animals is little studied. Here, we used 16S sequences obtained from clone libraries to characterise the faecal microbiota of Weddell seals breeding in McMurdo Sound and at White Island, Antarctica. Faecal bacterial communities were dominated by four phyla; Actinobacteria (20 %), Bacteroidetes (13 %), Firmicutes (23 %), and Proteobacteria (13 %). We also used automated ribosomal intergenic spacer analysis to examine the dispersal of bacteria between populations of Weddell seals breeding at White Island and in McMurdo Sound. The Weddell seals at White Island are isolated by the Ross Ice Shelf from the larger population of Weddell seals breeding in McMurdo Sound. We found that the faecal bacteria communities of the seals at White Island had lower diversity and that the community composition was significantly different compared with the seals in the McMurdo Sound area.     

Soil bacterial communities of different natural forest types in Northeast China

Background and aims

The types of natural forests have long been suggested to shape below-ground microbial communities in forest ecosystem. However, detailed information on the impressionable bacterial groups and the potential mechanisms of these influences are still missing. The present study aims to deepen the current understanding on the soil microbial communities under four typical forest types in Northeast Asia, and to reveal the environmental factors driving the abundance, diversity and composition of soil bacterial communities.

Methods

Four forest types from Changbai Nature Reserve, representing mixed conifer-broadleaf forest and its natural secondary forest, evergreen coniferous forest, and deciduous coniferous forest were selected for this study. Namely, Broadleaf-Korean pine mixed forest (BLKP), secondary Poplar-Birch forest (PB), Spruce-Fir forest (SF), and Larch forest (LA), respectively. Soil bacterial community was analyzed using bar-coded pyrosequencing. Nonmetric multidimensional scaling (NMDS) was used to illustrate the clustering of different samples based on both Bray-Curtis distances and UniFrac distances. The relationship between environmental variables and the overall community structure was analyzed using the Mantel test.

Results

The two mixed conifer-broadleaf forests (BLKP and PB) displayed higher total soil nutrients (organic carbon, nitrogen, and phosphorus) and soil pH, but a lower C/N ratio as compared to the two coniferous forests (SF and LA). The mixed conifer-broadleaf forests had higher alpha-diversity and had distinct bacterial communities from the coniferous forests. Soil texture and pH were found as the principle factors for shaping soil bacterial diversity and community composition. The two mixed conifer-broadleaf forests were associated with higher proportion of Acidobacteria, Verrucomicrobia, Bacteroidetes, and Chloroflexi. While the SF and LA forests were dominated by Proteobacteria and Gemmatimonadetes.

Conclusions

Different natural forest type each selects for distinct microbial communities beneath them, with mixed conifer-broadleaf forests being associated with the low-activity bacterial groups, and the coniferous forests being dominated by the so-called high-activity members. The differentiation of soil bacterial communities in natural forests are presumably mediated by the differentiation in terms of soil properties, and could be partially explained by the copiotroph/oligotroph ecological classification model and non-random co-occurrence patterns.     

Bacterial Community Assemblages Associated with the Phyllosphere,Dermosphere, and Rhizosphere of Tree Species of the Atlantic Forest are Host Taxon Dependent

Bacterial communities associated with tree canopies have been shown to be specific to their plant hosts, suggesting that plant species-specific traits may drive the selection of microbial species that comprise their microbiomes. To further examine the degree to which the plant taxa drive the assemblage of bacterial communities in specific plant microenvironments, we evaluated bacterial community structures associated with the phyllosphere, dermosphere, and rhizosphere of seven tree species representing three orders, four families and four genera of plants from a pristine Dense Ombrophilous Atlantic forest in Brazil, using a combination of PCR-DGGE of 16S rRNA genes and clone library sequencing. Results indicated that each plant species selected for distinct bacterial communities in the phyllosphere, dermosphere, and rhizosphere, and that the bacterial community structures are significantly related to the plant taxa, at the species, family, and order levels. Further characterization of the bacterial communities of the phyllosphere and dermosphere of the tree species showed that they were inhabited predominantly by species of Gammaproteobacteria, mostly related to Pseudomonas. In contrast, the rhizosphere bacterial communities showed greater species richness and evenness, and higher frequencies of Alphaproteobacteria and Acidobacteria Gp1. With individual tree species each selecting for their specific microbiomes, these findings greatly increase our estimates of the bacterial species richness in tropical forests and provoke questions concerning the ecological functions of the microbial communities that exist on different plant parts.     

The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.     

Role of PARP2 in DNA repair

The genome stability of higher eukaryotes depends largely on the functioning of the DNA repair systems. In turn, the precise regulation of each step of repair processes is necessary for the efficient DNA repair. Although most pathways of DNA repair have already been established, their regulation mechanisms require further investigation. Poly(ADP-ribose) polymerases (PARPs) are widely considered to be potential regulators of DNA repair. The role of the most prominent member of this protein family, i.e., PARP1, in DNA repair has been being intensively studied, while the literature data on participation in the repair processes of PARP2, the closest PARP1 homolog, are poorly summarized, although a great body of information concerning its participation in DNA repair has been accumulated. Using the PARP2-deficient model organisms and cell lines, their increased sensitivity to several DNA damaging agents was elucidated. The accumulation of PARP2 at the DNA damage sites in cells was shown. There are data that demonstrate the proteinprotein interaction of PARP2 with several proteins of the base excision repair/single-strand break repair and nonhomologous end joining. Most of the data on the PARP2 role were obtained in experiments with model organisms and cell lines; thus, it is difficult to elucidate the influence of PARP2 on specific processes in vivo. In this review, we tried to summarize data on the participation of PARP2 in the DNA repair processes, including our recent results.     

Bacterial Distribution Along a 50 °C Temperature Gradient Reveals a Parceled Out Hot Spring Environment

Understanding the distribution of bacteria is a major goal of microbial ecology which remains to be fully deciphered. In this study, a model 50 °C temperature gradient at a Northern Thailand hot spring was analyzed to determine how the bacterial communities were structured in the environment. Communities were examined through 16S rRNA gene amplification, denaturing gradient gel electrophoresis, and sequencing. The two major phyla, Cyanobacteria and Chloroflexi, showed characteristic distributions along the temperature gradient. Different clades were allocated at specific portions of the gradient. Comparisons of the bacterial communities along the temperature gradient showed sharp decreases of similarity at increasing temperature difference. Peaks of maximum richness were observed at 50 and 70 °C. This study contributes to explain how environmental conditions and microbial interactions can influence the distribution of specific bacterial clades and phyla shaping the structure of microbial communities in nature.     

PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences

Background

Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy.

Methodology/Principal Findings

A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI''s Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans.

Conclusions/Significance

Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans.     

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale “fill-and-draw” reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.     

Effects of experimental warming and nitrogen fertilization on soil microbial communities and processes of two subalpine coniferous species in Eastern Tibetan Plateau,China

Aim

This study aimed at predicting how sub-alpine coniferous ecosystems respond to global changes in the Eastern Tibetan Plateau by understanding soil microbial communities and activities, as well as variation in the quality and quantity of soil organic matter.

Methods

An experiment was conducted to examine soil microbial communities and their related soil processes in rhizospheric soil of two coniferous species that were exposed to two levels of temperature (unwarmed and infrared heater warming) and two levels of nitrogen (unfertilized and 25 g N m^?2 a^?1) from April 2007.

Results

Four-year night warming alone slightly affected the phospholipid fatty acid contents of the microbial community. However, the combination of nitrogen addition and soil warming significantly affected soil microbial composition while reducing the biomass of major microbial groups and the activities of most enzymes, especially in Abies faxoniana plots. The combination of warming and nitrogen addition increased soil labile C and N pools in Picea asperata plots and was beneficial for soil recalcitrant C, as well as for labile and total C and N pools in A. faxoniana plots.

Conclusion

Results indicated that future warming will slightly affect soil microbial communities and their related soil processes. However, warming combined with high nitrogen deposition will significantly constrain soil microbial biomass and enzyme activities, consequently increasing soil C and N pools in sub-alpine coniferous forests of this region.     

Does the Aboveground Herbivore Assemblage Influence Soil Bacterial Community Composition and Richness in Subalpine Grasslands?

Grassland ecosystems support large communities of aboveground herbivores that are known to directly and indirectly affect belowground properties such as the microbial community composition, richness, or biomass. Even though multiple species of functionally different herbivores coexist in grassland ecosystems, most studies have only considered the impact of a single group, i.e., large ungulates (mostly domestic livestock) on microbial communities. Thus, we investigated how the exclusion of four groups of functionally different herbivores affects bacterial community composition, richness, and biomass in two vegetation types with different grazing histories. We progressively excluded large, medium, and small mammals as well as invertebrate herbivores using exclosures at 18 subalpine grassland sites (9 per vegetation type). We assessed the bacterial community composition using terminal restriction fragment length polymorphism (T-RFLP) at each site and exclosure type during three consecutive growing seasons (2009–2011) for rhizosphere and mineral soil separately. In addition, we determined microbial biomass carbon (MBC), root biomass, plant carbon:nitrogen ratio, soil temperature, and soil moisture. Even though several of these variables were affected by herbivore exclusion and vegetation type, against our expectations, bacterial community composition, richness, or MBC were not. Yet, bacterial communities strongly differed between the three growing seasons as well as to some extent between our study sites. Thus, our study indicates that the spatiotemporal variability in soil microclimate has much stronger effects on the soil bacterial communities than the grazing regime or the composition of the vegetation in this high-elevation ecosystem.     

A meta‐analysis of temperature sensitivity as a microbial trait

Traits‐based approaches in microbial ecology provide a valuable way to abstract organismal interaction with the environment and to generate hypotheses about community function. Using macromolecular rate theory (MMRT), we recently identified that temperature sensitivity can be characterized as a distinct microbial trait. As temperature is fundamental in controlling biological reactions, variation in temperature sensitivity across communities, organisms, and processes has the potential to vastly improve understanding of microbial response to climate change. These microbial temperature sensitivity traits include the heat capacity (), temperature optimum (T_opt), and point of maximum temperature sensitivity (TS_max), each of which provide unique insights about organismal response to changes in temperature. In this meta‐analysis, we analyzed the distribution of these temperature sensitivity traits from bacteria, fungi, and mixed communities across a variety of biological systems (e.g., soils, oceans, foods, wastewater treatment plants) in order to identify commonalities in temperature responses across these diverse organisms and reaction rates. Our analysis of temperature sensitivity traits from over 350 temperature response curves reveals a wide distribution of temperature sensitivity traits, with T_opt and TS_max well within biological relevant temperatures. We find that traits vary significantly depending on organism type, microbial diversity, source environment, and biological process, with higher temperature sensitivity found in fungi than bacteria and in less diverse systems. Carbon dioxide production was found to be less temperature sensitive than denitrification, suggesting that changes in temperature will have a potentially larger impact on nitrogen‐related processes. As climate changes, these results have important implications for basic understanding of the temperature sensitivity of biological reactions and for ecological understanding of species’ trait distributions, as well as for improved treatment of temperature sensitivity in models.     

Effects of inorganic nanoparticles on viability and catabolic activities of Agrobacterium sp. PH-08 during biodegradation of dibenzofuran

This study investigated the cytotoxicity, genotoxicity, and growth inhibition effects of four different inorganic nanoparticles (NPs) such as aluminum (nAl), iron (nFe), nickel (nNi), and zinc (nZn) on a dibenzofuran (DF) degrading bacterium Agrobacterium sp. PH-08. NP (0–1,000 mg L^?1) -treated bacterial cells were assessed for cytotoxicity, genotoxicity, growth and biodegradation activities at biochemical and molecular levels. In an aqueous system, the bacterial cells treated with nAl, nZn and nNi at 500 mg L^?1 showed significant reduction in cell viability (30–93.6 %, p < 0.05), while nFe had no significant inhibition on bacterial cell viability. In the presence of nAl, nZn and nNi, the cells exhibited elevated levels of reactive oxygen species (ROS), DNA damage and cell death. Furthermore, NP exposure showed significant (p < 0.05) impairment in DF and catechol biodegradation activities. The reduction in DF biodegradation was ranged about 71.7–91.6 % with single NPs treatments while reached up to 96.3 % with a mixture of NPs. Molecular and biochemical investigations also clearly revealed that NP exposure drastically affected the catechol-2,3-dioxygenase activities and its gene (c23o) expression. However, no significant inhibition was observed in nFe treatment. The bacterial extracellular polymeric materials and by-products from DF degradation can be assumed as key factors in diminishing the toxic effects of NPs, especially for nFe. This study clearly demonstrates the impact of single and mixed NPs on the microbial catabolism of xenobiotic-degrading bacteria at biochemical and molecular levels. This is the first study on estimating the impact of mixed NPs on microbial biodegradation.     

Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil

Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial community in soil. The current study provides information on the response of soil bacterial, archaeal and fungal communities during the degradation of three priority pollutants and contributes to a knowledge base that can inform the development of effective bioremediation strategies for contaminated sites.     

Responsiveness of cardiometabolic-related microbiota to diet is influenced by host genetics

Intestinal microbial community structure is driven by host genetics in addition to environmental factors such as diet. In comparison with environmental influences, the effect of host genetics on intestinal microbiota, and how host-driven differences alter host metabolism is unclear. Additionally, the interaction between host genetics and diet, and the impact on the intestinal microbiome and possible down-stream effect on host metabolism is not fully understood, but represents another aspects of inter-individual variation in disease risk. The objectives of this study were to investigate how diet and genetic background shape microbial communities, and how these diet- and genetic-driven microbial differences relate to cardiometabolic phenotypes. To determine these effects, we used the 8 progenitor strains of the collaborative cross/diversity outbred mapping panels (C57BL/6J, A/J, NOD/ShiLtJ, NZO/HILtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ). 16s rRNA profiling of enteric microbial communities in addition to the assessment of phenotypes central to cardiometabolic health was conducted under baseline nutritional conditions and in response to diets varying in atherogenic nutrient (fat, cholesterol, cholic acid) composition. These studies revealed strain-driven differences in enteric microbial communities which were retained with dietary intervention. Diet–strain interactions were seen for a core group of cardiometabolic-related microbial taxa. In conclusion, these studies highlight diet and genetically regulated cardiometabolic-related microbial taxa. Furthermore, we demonstrate the progenitor model is useful for nutrigenomic-based studies and screens seeking to investigate the interaction between genetic background and the phenotypic and microbial response to diet.     

Antimicrobial and Antibiofilm Activity of Mauriporin,a Multifunctional Scorpion Venom Peptide

Many pathogenic free living and biofilm forming bacterial organisms can cause serious infections to humans that could consequently have devastating effects on human health. A significant number of these microbial organisms are resistant to almost all known conventional antibiotics and the ability of some these strains to form sessile communities of biofilms increases the resistance ability of bacteria to antibiotic treatment. Global research is currently focused on finding novel therapies to counteract the threat of bacterial and biofilm infections rather than using conventional antibiotics. Mauriporin, a novel cationic α-helical peptide identified from the venom derived cDNA library of the scorpion Androctonus mauritanicus was reported to display selective cytotoxic and anti-proliferative activity against prostate cancer cell lines. In the present study, we investigated the antimicrobial and antibiofilm activities of Mauriporin. Our results show that Mauriporin displays potent antimicrobial activities against a range of Gram-positive and Gram-negative planktonic bacteria with MIC values in the range 5 µM to 10 µM. Mauriporin was also able to prevent Pseudomonas aeruginosa biofilm formation while showing weak hemolytic activity towards human erythrocytes. Studies on the mechanism of action of Mauriporin revealed that the peptide is probably inducing bacterial cell death through membrane permeabilization determined by the release of β-galactosidase enzyme from peptide treated Escherichia coli cells. Moreover, DNA binding studies found that Mauriporin can cause potent binding to intracellular DNA. All these results indicate that Mauriporin has a considerable potential for therapeutic application as a novel drug candidate for eradicating bacterial infections.     

Impact of single-cell genomics and metagenomics on the emerging view of extremophile “microbial dark matter”

Despite >130 years of microbial cultivation studies, many microorganisms remain resistant to traditional cultivation approaches, including numerous candidate phyla of bacteria and archaea. Unraveling the mysteries of these candidate phyla is a grand challenge in microbiology and is especially important in habitats where they are abundant, including some extreme environments and low-energy ecosystems. Over the past decade, parallel advances in DNA amplification, DNA sequencing and computing have enabled rapid progress on this problem, particularly through metagenomics and single-cell genomics. Although each approach suffers limitations, metagenomics and single-cell genomics are particularly powerful when combined synergistically. Studies focused on extreme environments have revealed the first substantial genomic information for several candidate phyla, encompassing putative acidophiles (Parvarchaeota), halophiles (Nanohaloarchaeota), thermophiles (Acetothermia, Aigarchaeota, Atribacteria, Calescamantes, Korarchaeota, and Fervidibacteria), and piezophiles (Gracilibacteria). These data have enabled insights into the biology of these organisms, including catabolic and anabolic potential, molecular adaptations to life in extreme environments, unique genomic features such as stop codon reassignments, and predictions about cell ultrastructure. In addition, the rapid expansion of genomic coverage enabled by these studies continues to yield insights into the early diversification of microbial lineages and the relationships within and between the phyla of Bacteria and Archaea. In the next 5 years, the genomic foliage within the tree of life will continue to grow and the study of yet-uncultivated candidate phyla will firmly transition into the post-genomic era.     

Microbial and metabolic characterization of a denitrifying phosphorus-uptake/side stream phosphorus removal system for treating domestic sewage

In this study, an advanced wastewater treatment process, the denitrifying phosphorus/side stream phosphorus removal system (DPR-Phostrip), was developed for the purpose of enhancing denitrifying phosphorus removal. The enrichment of denitrifying phosphorus-accumulating organisms (DPAOs) and the microbial community structure of DPR-Phostrip were evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and the metabolic activity of seed sludge and activated sludge collected after 55 days of operation were evaluated by Biolog? analysis. This experimental study of DPR-Phostrip operation showed that nutrients were removed effectively, and denitrifying phosphorus removal was observed during the pre-anoxic period. PCR-DGGE analysis indicated that DPR-Phostrip supported DPAO growth while inhibiting PAOs and GAOs. The major dominant species in DPR-Phostrip were Bacteroidetes bacterium, Saprospiraceae bacterium, and Chloroflexi bacterium. Moreover, the functional diversity indices calculated on the basis of Biolog analysis indicated that DPR-Phostrip had almost no effect on microbial community diversity but was associated with a shift in the dominant species, which confirms the results of the PCR-DGGE analysis. The results for average well color development, calculated via Biolog analysis, showed that DPR-Phostrip had a little impact on the metabolic activity of sludge. Further principal component analysis suggested that the ability to utilize low-molecular-weight organic compounds was reduced in DPR-Phostrip.     

Genome stability: recent insights in the topoisomerase reverse gyrase and thermophilic DNA alkyltransferase

Repair and defence of genome integrity from endogenous and environmental hazard is a primary need for all organisms. Natural selection has driven the evolution of multiple cell pathways to deal with different DNA damaging agents. Failure of such processes can hamper cell functions and induce inheritable mutations, which in humans may cause cancerogenicity or certain genetic syndromes, and ultimately cell death. A special case is that of hyperthermophilic bacteria and archaea, flourishing at temperatures higher than 80 °C, conditions that favor genome instability and thus call for specific, highly efficient or peculiar mechanisms to keep their genome intact and functional. Over the last few years, numerous studies have been performed on the activity, function, regulation, physical and functional interaction of enzymes and proteins from hyperthermophilic microorganisms that are able to bind, repair, bypass damaged DNA, or modify its structure or conformation. The present review is focused on two enzymes that act on DNA catalyzing unique reactions: reverse gyrase and DNA alkyltransferase. Although both enzymes belong to evolutionary highly conserved protein families present in organisms of the three domains (Eucarya, Bacteria and Archaea), recently characterized members from hyperthermophilic archaea show both common and peculiar features.