Microbial Biology Research Articles

Terabase-Scale Coassembly of a Tropical Soil Microbiome.

Petabases of environmental metagenomic data are publicly available, presenting an opportunity to characterize complex environments and discover novel lineages of life. Metagenome coassembly, in which many metagenomic samples from an environment are simultaneously analyzed to infer the underlying genomes' sequences, is an essential tool for achieving this goal. We applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 terabases (Tbp) of metagenome data from a tropical soil in the Luquillo Experimental Forest (LEF), Puerto Rico. The resulting coassembly yielded 39 high-quality (>90% complete, <5% contaminated, with predicted 23S, 16S, and 5S rRNA genes and ≥18 tRNAs) metagenome-assembled genomes (MAGs), including two from the candidate phylum Eremiobacterota. Another 268 medium-quality (≥50% complete, <10% contaminated) MAGs were extracted, including the candidate phyla Dependentiae, Dormibacterota, and Methylomirabilota. In total, 307 medium- or higher-quality MAGs were assigned to 23 phyla, compared to 294 MAGs assigned to nine phyla in the same samples individually assembled. The low-quality (<50% complete, <10% contaminated) MAGs from the coassembly revealed a 49% complete rare biosphere microbe from the candidate phylum FCPU426 among other low-abundance microbes, an 81% complete fungal genome from the phylum Ascomycota, and 30 partial eukaryotic MAGs with ≥10% completeness, possibly representing protist lineages. A total of 22,254 viruses, many of them low abundance, were identified. Estimation of metagenome coverage and diversity indicates that we may have characterized ≥87.5% of the sequence diversity in this humid tropical soil and indicates the value of future terabase-scale sequencing and coassembly of complex environments. IMPORTANCE Petabases of reads are being produced by environmental metagenome sequencing. An essential step in analyzing these data is metagenome assembly, the computational reconstruction of genome sequences from microbial communities. "Coassembly" of metagenomic sequence data, in which multiple samples are assembled together, enables more complete detection of microbial genomes in an environment than "multiassembly," in which samples are assembled individually. To demonstrate the potential for coassembling terabases of metagenome data to drive biological discovery, we applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 Tbp of reads from a humid tropical soil environment. The resulting coassembly, its functional annotation, and analysis are presented here. The coassembly yielded more, and phylogenetically more diverse, microbial, eukaryotic, and viral genomes than the multiassembly of the same data. Our resource may facilitate the discovery of novel microbial biology in tropical soils and demonstrates the value of terabase-scale metagenome sequencing.

The Extracellular Matrix of Yeasts: A Key Player in the Microbial Biology Change of Paradigm.

Microbes are traditionally regarded as planktonic organisms, individual cells that live independently from each other. Although this is true, microbes in nature mostly live within large multi-species communities forming complex ecosystems. In these communities, microbial cells are held together and organised spatially by an extracellular matrix (ECM). Unlike the ECM from the tissues of higher eukaryotes, microbial ECM, mostly that of yeasts, is still poorly studied. However, microbial biofilms are a serious cause for concern, for being responsible for the development of nosocomial infections by pharmacological drugs-resistant strains of pathogens, or for critically threatening plant health and food security under climate change. Understanding the organization and behaviour of cells in biofilms or other communities is therefore of extreme importance. Within colonies or biofilms, extremely large numbers of individual microbial cells adhere to inert surfaces or living tissues, differentiate, die or multiply and invade adjacent space, often following a 3D architectural programme genetically determined. For all this, cells depend on the production and secretion of ECM, which might, as in higher eukaryotes, actively participate in the regulation of the group behaviour. This work presents an overview of the state-of-the-art on the composition and structure of the ECM produced by yeasts, and the inherent physicochemical properties so often undermined, as well as the available information on its production and delivery pathways.

Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species.

The phage T7 RNA polymerase (RNAP) and lysozyme form the basis of the widely used pET expression system for recombinant expression in the biotechnology field and as a tool in microbial synthetic biology. Attempts to transfer this genetic circuitry from Escherichia coli to non-model bacterial organisms with high potential have been restricted by the cytotoxicity of the T7 RNAP in the receiving hosts. We here explore the diversity of T7-like RNAPs mined directly from Pseudomonas phages for implementation in Pseudomonas species, thus relying on the co-evolution and natural adaptation of the system towards its host. By screening and characterizing different viral transcription machinery using a vector-based system in P. putida., we identified a set of four non-toxic phage RNAPs from phages phi15, PPPL-1, Pf-10, and 67PfluR64PP, showing a broad activity range and orthogonality to each other and the T7 RNAP. In addition, we confirmed the transcription start sites of their predicted promoters and improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition. This set of viral RNAPs expands the adaption of T7-inspired circuitry towards Pseudomonas species and highlights the potential of mining tailored genetic parts and tools from phages for their non-model host.

Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri.

Killifish are emerging as a new laboratory system in which to study a range of questions, from the genetic basis of embryo dormancy to life history trait evolution, age-dependent neurodegeneration, and the connection between microbial community structure and biology of aging. Over the past decade, advances in high-throughput sequencing have helped uncover the vast diversity of microbial communities present in environmental samples and on host epithelia. Here, we describe an optimized protocol to study the taxonomic composition of intestinal and fecal microbiota in laboratory-raised as well as natural killifish populations and provide comprehensive step-by-step instructions for tissue sampling, high-throughput genomic DNA extraction, and the generation of 16S V3V4 rRNA and 16S V4 rRNA gene libraries.

House Flies Are Underappreciated Yet Important Reservoirs and Vectors of Microbial Threats to Animal and Human Health.

House flies are well recognized as filth-associated organisms and public nuisances. House flies create sanitation issues when they bridge the gap between microbe-rich breeding environments and animal/human habitations. Numerous scientific surveys have demonstrated that house flies harbor bacterial pathogens that pose a threat to humans and animals. More extensive and informative surveys incorporating next-generation sequencing technologies have shown that house fly carriage of pathogens and harmful genetic elements, such as antimicrobial resistance genes, is more widespread and dangerous than previously thought. Further, there is a strong body of research confirming that flies not only harbor but also transmit viable, and presumably infectious, bacterial pathogens. Some pathogens replicate and persist in the fly, permitting prolonged shedding and dissemination. Finally, although the drivers still have yet to be firmly determined, the potential range of dissemination of flies and their associated pathogens can be extensive. Despite this evidence, the house flies' role as reservoirs, disseminators, and true, yet facultative, vectors for pathogens have been greatly underestimated and underappreciated. In this review, we present key studies that bolster the house fly's role both an important player in microbial ecology and population biology and as transmitters of microbial threats to animal and human health.

Review Article on Microbiology

Microbiology is the study of microorganisms biological entities too small to be seen with the unaided eye. Most major advances in microbiology have occurred within the past 150 years, and several important subdisciplines of microbiology have developed during this time, including microbial ecology, molecular biology, immunology, industrial microbiology and biotech- nology. Microorganisms of various types exist in all three domains of life (the Bacteria, Archaea and Eukarya), and they are by far the most abundant life forms on Earth. Microscopic biological agents include bacteria, archaea, protists (protozoa and algae), fungi, parasitic worms (helminths) and viruses. Although a small percentage of microorganisms are harmful to certain plants and animals and may cause serious disease in humans, the vast majority of microorganisms provide beneficial services, such as assisting in water purification and the production of certain foods, and many are essential for the proper functioning of Earth's ecosystems. Molecular biology has revolutionized our understanding of the diversity, function, and community structure of marine microorganisms. Increasingly, tools and techniques derived from the biomedical diagnostics and research industries are used in parallel with sensors that characterize the physical, chemical, and optical properties of ocean waters.

Review on Microbiology and Molecular Biology

Microbiology is the study of microorgan- isms biological entities too small to be seen with the unaided eye. Most major advances in microbiology have occurred within the past 150 years, and several important subdisciplines of microbiology have developed during this time, including microbial ecology, molecular biology, immunology, industrial microbiology and biotech- nology. Microorganisms of various types exist in all three domains of life (the Bacteria, Archaea and Eukarya), and they are by far the most abundant life forms on Earth. Microscopic biological agents include bacteria, archaea, protists (protozoa and algae), fungi, parasitic worms (helminths) and viruses. Although a small percentage of microor- ganisms are harmful to certain plants and animals and may cause serious disease in humans, the vast majority of microorganisms provide beneficial services, such as assisting in water purification and the production of certain foods, and many are essential for the proper functioning of Earth's ecosystems. Molecular biology has revolutionized our understanding of the diversity, function, and community structure of marine microorganisms. Increasingly, tools and techniques derived from the biomedical diagnostics and research industries are used in parallel with sensors that characterize the physical, chemical, and optical properties of ocean waters.

Role of Bacteriophages in the Evolution of Pathogenic Vibrios and Lessons for Phage Therapy.

Viruses of bacteria, i.e., bacteriophages (or phages for short), were discovered over a century ago and have played a major role as a model system for the establishment of the fields of microbial genetics and molecular biology. Despite the relative simplicity of phages, microbiologists are continually discovering new aspects of their biology including mechanisms for battling host defenses. In turn, novel mechanisms of host defense against phages are being discovered at a rapid clip. A deeper understanding of the arms race between bacteria and phages will continue to reveal novel molecular mechanisms and will be important for the rational design of phage-based prophylaxis and therapies to prevent and treat bacterial infections, respectively. Here we delve into the molecular interactions of Vibrio species and phages.

The enhanced expression of genes encoding diguanylate cyclases under cold stress contributes to the adhesion and biofilm formation of Shewanella putrefaciens WS13.

Shewanella putrefaciens is a special spoilage bacterium of seafood during cold storage, which is easy to form biofilm and bring serious hazard to the seafood quality. Life cycle of biofilm starts after bacterial adhesion, which is essential for the formation and development of biofilm. As a ubiquitous second messenger in bacteria, c-di-GMP regulates the conversion between bacterial planktonic state and biofilm state. In this study, the adhesion and biofilm formation of S. putrefaciens WS13 under 4°C were compared to those under 30°C. Atom force microscope and scanning electron microscope were used to study the bacterial adhesion. Biofilm was analyzed by Fourier transform infrared spectroscopy, Bradford assay and phenol-sulfuric acid method. High-performance liquid chromatographic-tandem mass spectrometric and quantitative real-time PCR were applied to study c-di-GMP level and genes encoding diguanylate cyclases in cells, respectively. Results showed that the swarming mobility of S. putrefaciens WS13 was weaker under 4°C, however, the adhesive force under 4°C was 4-5 times higher than that under 30°C. Biofilm biomass, extracellular polysaccharides and extracellular proteins were 2.5 times, 3 times, and 1.6 times more than those under 30°C, respectively, but biofilm composition formed under both temperatures were similar. c-di-GMP level in S. putrefaciens WS13 under 30°C was no more than half of that in the corresponding growth stage under 4°C. Quantitative real-time PCR analysis also showed that the expression of genes encoding diguanylate cyclases were significantly enhanced under 4°C than that under 30°C. S. putrefaciens WS13 adapted to the cold stress by enhancing the expression of genes encoding diguanylate cyclases to promote bacterial adhesion and biofilm formation. This study provides a theoretical foundation for the research on the cold adaptation mechanism of specific spoilage bacteria of seafood based on c-di-GMP, and also provides a new idea to control seafood quality from the perspective of microbial molecular biology.

Louis Pasteur, molecular dissymmetry, therapeutic chemistry and neuropharmacology.

Louis Pasteur is celebrated as the founding father of microbiology. But he was a chemist by training and discovered molecular dissymmetry experimentally. All his life, his constant preoccupation will be to apply the method and strategies of the fundamental sciences to living processes, "from the molecule to the brain". His fundamental aim will be, beyond the biology of microbes, the chemistry of life, a disposition which signs the originality of his work. More unexpectedly, Pasteur was at the origin of therapeutic chemistry-which his successors, and especially Daniel Bovet, brilliantly illustrated at the Pasteur Institute and which they would pursue with the pharmacology of the nervous system or "neuropharmacology".

Microbial production of the plant-derived fungicide physcion

Physcion is a characteristic component of the traditional herb rhubarb with diverse pharmacological activities that has been commercially approved as an herbal fungicide. Nevertheless, its extremely low contents, costly purification procedure and geographically restricted planting severely hinder its application. Here, a cell factory was constructed in the filamentous fungus Aspergillus terreus for physcion production via microbial fermentation by integrating a pathway-modified emodin accumulation module and a position-selective emodin methylation module. Specifically, 1.71 g/L emodin accumulated when the transcriptional activator GedR and the emodin-1-OH-O-methyltransferase GedA in the geodin biosynthetic pathway were overexpressed and knocked out, respectively. Subsequently, potential emodin-3-OH-O-methyltransferase candidates were enzymatically screened in vitro and introduced into the emodin-accumulating mutant in vivo to generate a physcion-producing strain showing the highest titre of 6.3 g/L in fed-batch fermentation. Thus, our study provides an alternative strategy for the highly efficient, economical production of physcion and a representative example for microbial synthetic biology.

Quorum Sensing and Antimicrobial Production Orchestrate Biofilm Dynamics in Multispecies Bacterial Communities.

Microbial interactions are often mediated by diffusible small molecules, including secondary metabolites, that play roles in cell-to-cell signaling and inhibition of competitors. Biofilms are often "hot spots" for high concentrations of bacteria and their secondary metabolites, which make them ideal systems for the study of small-molecule contributions to microbial interactions. Here, we use a five-member synthetic community consisting of Roseobacteraceae representatives to investigate the role of secondary metabolites on microbial biofilm dynamics. One synthetic community member, Rhodobacterales strain Y4I, possesses two acylated homoserine lactone (AHL)-based cell-to-cell signaling systems (pgaRI and phaRI) as well as a nonribosomal peptide synthase gene (igi) cluster that encodes the antimicrobial indigoidine. Through serial substitution of Y4I with mutants deficient in single signaling molecule pathways, the contribution of these small-molecule systems could be assessed. As secondary metabolite production is dependent upon central metabolites, the influence of growth substrate (i.e., complex medium versus defined medium with a single carbon substrate) on these dynamics was also considered. Depending on the Y4I mutant genotype included, community dynamics ranged from competitive to cooperative. The observed interactions were mostly competitive in nature. However, the community harboring a Y4I variant that was both impaired in quorum sensing (QS) pathways and unable to produce indigoidine (pgaR variant) shifted toward more cooperative interactions over time. These cooperative interactions were enhanced in the defined growth medium. The results presented provide a framework for deciphering complex, small-molecule-mediated interactions that have broad application to microbial biology. IMPORTANCE Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities. Few studies have examined the impact of secondary metabolites, such as cell-to-cell signaling and antimicrobial production, on marine microbial biofilm community structure. Here, we assessed the impact of secondary metabolites on microbial interactions using a synthetic, five-member Roseobacteraceae community by measuring species composition and biomass production during biofilm growth. We present evidence that secondary metabolites influence social behaviors within these multispecies microbial biofilms, thereby improving understanding of bacterial secondary metabolite production influence on social behaviors within marine microbial biofilm communities.

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity

BackgroundGlucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides.ResultsIn this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides.ConclusionsTwo highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

Photoantimicrobials in agriculture.

Classical approaches for controlling plant pathogens may be impaired by the development of pathogen resistance to chemical pesticides and by limited availability of effective antimicrobial agents. Recent increases in consumer awareness of and/or legislation regarding environmental and human health, and the urgent need to improve food security, are driving increased demand for safer antimicrobial strategies. Therefore, there is a need for a step change in the approaches used for controlling pre- and post-harvest diseases and foodborne human pathogens. The use of light-activated antimicrobial substances for the so-called antimicrobial photodynamic treatment is known to be effective not only in a clinical context, but also for use in agriculture to control plant-pathogenic fungi and bacteria, and to eliminate foodborne human pathogens from seeds, sprouted seeds, fruits, and vegetables. Here, we take a holistic approach to review and re-evaluate recent findings on: (i) the ecology of naturally-occurring photoantimicrobials, (ii) photodynamic processes including the light-activated antimicrobial activities of some plant metabolites, and (iii) fungus-induced photosensitization of plants. The inhibitory mechanisms of both natural and synthetic light-activated substances, known as photosensitizers, are discussed in the contexts of microbial stress biology and agricultural biotechnology. Their modes-of-antimicrobial action make them neither stressors nor toxins/toxicants (with specific modes of poisonous activity), but a hybrid/combination of both. We highlight the use of photoantimicrobials for the control of plant-pathogenic fungi and quantify their potential contribution to global food security.

Rehabilitation of the 'bad guys' for biocontrol applications.

Environmental MicrobiologyVolume 25, Issue 1 p. 97-101 Crystal Ball Rehabilitation of the ‘bad guys’ for biocontrol applications Leo Eberl, Corresponding Author Leo Eberl [email protected] orcid.org/0000-0002-7241-0864 Department of Plant and Microbial biology, University of Zurich, Zurich, Switzerland Correspondence Leo Eberl, Department of Plant and Microbial biology, University of Zurich, Zurich, Switzerland. Email: [email protected]Search for more papers by this author Leo Eberl, Corresponding Author Leo Eberl [email protected] orcid.org/0000-0002-7241-0864 Department of Plant and Microbial biology, University of Zurich, Zurich, Switzerland Correspondence Leo Eberl, Department of Plant and Microbial biology, University of Zurich, Zurich, Switzerland. Email: [email protected]Search for more papers by this author First published: 18 September 2022 https://doi.org/10.1111/1462-2920.16216Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume25, Issue1January 2023Pages 97-101 RelatedInformation

Microbes of traditional fermentation processes as synthetic biology chassis to tackle future food challenges.

Microbial diversity is magnificent and essential to almost all life on Earth. Microbes are an essential part of every human, allowing us to utilize otherwise inaccessible resources. It is no surprise that humans started, initially unconsciously, domesticating microbes for food production: one may call this microbial domestication 1.0. Sourdough bread is just one of the miracles performed by microbial fermentation, allowing extraction of more nutrients from flour and at the same time creating a fluffy and delicious loaf. There are a broad range of products the production of which requires fermentation such as chocolate, cheese, coffee and vinegar. Eventually, with the rise of microscopy, humans became aware of microbial life. Today our knowledge and technological advances allow us to genetically engineer microbes - one may call this microbial domestication 2.0. Synthetic biology and microbial chassis adaptation allow us to tackle current and future food challenges. One of the most apparent challenges is the limited space on Earth available for agriculture and its major tolls on the environment through use of pesticides and the replacement of ecosystems with monocultures. Further challenges include transport and packaging, exacerbated by the 24/7 on-demand mentality of many customers. Synthetic biology already tackles multiple food challenges and will be able to tackle many future food challenges. In this perspective article, we highlight recent microbial synthetic biology research to address future food challenges. We further give a perspective on how synthetic biology tools may teach old microbes new tricks, and what standardized microbial domestication could look like.

Methylothon: a Versatile Course-Based High School Research Experience in Microbiology and Bioinformatics with Pink Bacteria.

ABSTRACTMethylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here, we present an overview of the module’s learning goals, describe course resources (available for public use at http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

Plants for human health: Almost gone but will not be forgotten

Using plants to develop therapy or treatment in humans seems to be a potential way to improve patient outcomes. The continuum of plant genetics changing into foods that improve health is an applicable strategy at this present. Horticultural Science, microbial biology, bioprocessing, and nutrition sciences require during the process. Therefore, the role of healthcare professionals including medical doctors, pharmacists, and nurses is needed to discover the benefits and process them into usable objects. Establishing coordinated efforts in the evolution of the resources to address health issues and ensure a successful way with institute initiatives program. Also, clarifying and delivering information to the public can be very useful to counteract the symptoms. In addition, a need for translation research along with innovative technology will reach the objective of this issue.

Microbial fingerprinting techniques and their role in the remediation of environmental pollution

Environmental pollution has increased in recent decades as a result of enhanced anthropogenic activities, poor farming techniques, and industrial growth. In the elimination of resistant environmental toxins (metals), bioremediation has been found as a desirable and efficient approach to environmental safety. Genomics, proteomics, metagenomics, metabolomics, transcriptomics, along with multidisciplinary approaches are used to characterize the function, metabolism, and composition of microbes, as well as to elucidate the environment based on changes in microbes, which has significant and directing relevance in environmental management, monitoring, and repair. Molecular microbiology approaches, among other microbial biodegradation techniques, have transformed microbial biotechnology, resulting in high-throughput and quick methods for culture-independent evaluation and utilization of bacteria existing in contaminated sites. Pollutants existing in contaminated locations, whether inorganic or organic, can disrupt the ecosystem by harming the fauna and flora. Because of its comparably high efficacy and safety, the microbial molecular biology technique might greatly increase the efficiency of naturally existing microbes for field bioremediation. Recent approaches with numerous techniques, including polymerase chain reaction (PCR), denaturating gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), terminal restriction fragment length polymorphism (TRFLP), rRNA intergenic spacer analysis (RISA), and single-strand conformational polymorphism (SSCP) can be used selectively in studies of microbial ecology and flora have supplied exceptional information about microbial communities and their function in bio-remediating environmental pollution. This review provides an overview of the uses of microbial fingerprinting approaches in the bioremediation of various contaminants in environmental matrices, as well as an explanation of current breakthroughs in such techniques' applications.

Hypoxia promotes cytolytic activity of CD8 T cells and pathogenesis in cutaneous leishmaniasis

Abstract Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and the most common form of the disease is cutaneous leishmaniasis (CL). A fundamental question in CL is what regulates the development of severe disease, information that is critical to develop therapies to ameliorate disease. In a series of studies, we demonstrated CD8 T cell-dependent cytotoxicity as the main inducer of immunopathology in CL. This result was unexpected since IFN-γ production by CD8 T cells plays a protective role by promoting pathogen elimination. To resolve this paradox, we studied the CD8 T cells in different anatomic sites and found that the effector function of CD8 T cells in CL depends on their location: while CD8 T cells are cytotoxic (GzmB+) and produce little IFN-γ in leishmania lesions, CD8 T cells in the draining lymph nodes (dLN) have the opposite profile. Importantly, GzmB− CD8 T cells from dLN quickly upregulate GzmB after injection into CL lesions. By transcriptional profiling, we found that CD8 T cells in lesions and not dLN have a hypoxic signature. In vivo, we observed that leishmania lesions are hypoxic using the Oxyphor G4 oxygen probe and pimonidazole staining. In vitro, we found that induction of hypoxia was sufficient to convert GzmB− into GzmB+ CD8 T cells. In vivo, blocking the dimerization of HIF-α, a master regulator of hypoxia, with acriflavine decreased lesion development in mice. Transcriptional profiling in patients showed that hypoxia is a signature of human CL and correlates with GZMB expression. Together, our results suggest that the hypoxic microenvironment of leishmania lesions alter the function of CD8 T cells and convert protective CD8 T cells into pathogenic cytotoxic T cells. Supported by Host Defense and Microbial Biology (OSU)