[ { "id": "authors:cq8bw-zsx24", "collection": "authors", "collection_id": "cq8bw-zsx24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170714-110726557", "type": "book_section", "title": "Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT)", "book_title": "Hydrocarbon and Lipid Microbiology Protocols: Single-Cell and Single-Molecule Methods", "author": [ { "family_name": "Hatzenpichler", "given_name": "Roland", "orcid": "0000-0002-5489-3444", "clpid": "Hatzenpichler-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "contributor": [ { "family_name": "McGenity", "given_name": "Terry J.", "clpid": "McGenity-T-J" }, { "family_name": "Timmis", "given_name": "Kenneth N.", "clpid": "Timmis-K-N" }, { "family_name": "Nogales", "given_name": "Balbina", "clpid": "Nogales-B" } ], "abstract": "Bioorthogonal noncanonical amino acid tagging (BONCAT) is a recently developed method for studying microbial in situ activity. This technique is based on the in vivo incorporation of artificial amino acids that carry modifiable chemical tags into newly synthesized proteins. BONCAT has been demonstrated to be effective in labeling the proteomes of a wide range of taxonomically and physiologically distinct Archaea and bacteria without resulting in preferential synthesis or degradation of proteins. After chemical fixation of cells, surrogate-containing proteins can be detected by whole-cell fluorescence staining using azide-alkyne click chemistry. When used in conjunction with rRNA-targeted fluorescence in situ hybridization (FISH), BONCAT allows the simultaneous taxonomic identification of a microbial cell and its translational activity. Rather than studying the bulk proteome, BONCAT is able to specifically target proteins that have been expressed in reaction to an experimental condition. BONCAT-FISH thus provides researchers with a selective, sensitive, fast, and inexpensive fluorescence microscopy technique for studying microbial in situ activity on an individual cell level.\n\nThis protocol provides a detailed description of how to design and perform BONCAT experiments using two different bioorthogonal amino acids, l-azidohomoalanine (AHA) and l-homopropargylglycine (HPG), which are both surrogates of l-methionine. It illustrates how incorporation of these noncanonical amino acids into new proteins can be detected via copper-catalyzed or strain-promoted azide-alkyne click chemistry and outlines how the visualization of translational activity can be combined with the taxonomic identification of cells via FISH. Last, the protocol discusses potential problems that might be encountered during BONCAT studies and how they can be overcome.", "doi": "10.1007/8623_2015_61", "isbn": "978-3-662-49129-4", "publisher": "Springer", "place_of_publication": "Berlin", "publication_date": "2015-04-04", "pages": "145-157" }, { "id": "authors:88swb-smm85", "collection": "authors", "collection_id": "88swb-smm85", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20131002-134144694", "type": "book_section", "title": "Whole Cell Immunomagnetic Enrichment of Environmental Microbial Consortia Using rRNA-Targeted Magneto-FISH", "book_title": "Microbial Metagenomics, Metatranscriptomics, and Metaproteomics", "author": [ { "family_name": "Trembath-Reichert", "given_name": "Elizabeth", "orcid": "0000-0002-3979-8676", "clpid": "Trembath-Reichert-E" }, { "family_name": "Green-Saxena", "given_name": "Abigail", "orcid": "0000-0002-8502-6589", "clpid": "Saxena-A-G" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "abstract": "Magneto-FISH, in combination with metagenomic techniques, explores the middle\nground between single-cell analysis and complex community characterization in bulk\nsamples to better understand microbial partnerships and their roles in ecosystems. The\nMagneto-FISH method combines the selectivity of catalyzed reporter deposition\u2013\nfluorescence in situ hybridization (CARD\u2013FISH) with immunomagnetic capture to provide\ntargeted molecular and metagenomic analysis of co-associated microorganisms in\nthe environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups\nassociated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as\nwell as provided insight into their physiological potential using metagenomics. Here,\nwe demonstrate the utility of this method for capturing aggregated consortia using\na series of nested oligonucleotide probes of differing specificity designed to target\neither the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA\nand mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for largeand\nsmall-volume samples and evaluates the strengths and limitations of this method\npredominantly focusing on (1) the relationship between FISH probe specificity and sample\nselectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples,\nand (3) suggestions for adapting the Magneto-FISH method for other microbial systems,\nincluding potential for single-cell recovery.", "doi": "10.1016/B978-0-12-407863-5.00002-2", "isbn": "9780124078635", "publisher": "Academic Press", "publication_date": "2013", "pages": "21-44" }, { "id": "authors:n7g2e-cr643", "collection": "authors", "collection_id": "n7g2e-cr643", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121106-134254144", "type": "book_section", "title": "Molecular biology's contributions to geobiology", "book_title": "Fundamentals of Geobiology", "author": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Reysenbach", "given_name": "Anna-Louise", "clpid": "Reysenbach-A-L" } ], "contributor": [ { "family_name": "Knoll", "given_name": "Andrew H.", "clpid": "Knoll-A-H" }, { "family_name": "Canfield", "given_name": "Donald E.", "clpid": "Canfield-D-E" }, { "family_name": "Konhauser", "given_name": "Kurt O.", "clpid": "Konhauser-K-O" } ], "abstract": "On August 7, 1996, US President Bill Clinton held a press conference to announce the possibility that the Allan Hills 84001 meteorite might provide insight into ancient life on Mars. With soaring rhetoric, he declared: 'Today, rock 84001 speaks to us across all those billions of years and millions of miles. It speaks of the possibility of life. If this discovery is confirmed, it will surely be one of the most stunning insights into our universe that science has ever uncovered. Its implications are as far-reaching and awe-inspiring as can be imagined. Even as it promises answers to some of our oldest questions, it poses still others even more fundamental.' Shortly thereafter, NASA expanded its support for astro-and geobiological research, which marked the beginning of a renaissance in geobiology. Seemingly overnight, geobiology was transformed from a somewhat arcane discipline to a glamorous field that promised to reveal the secrets of life. While today, most geobiologists would agree that the evidence for past life in AH84001 is inconclusive at best, and find the hype surrounding its discovery to be comical, nonetheless, the excitement it engendered has had a long-lasting and positive impact on our science. The enduring consequence of Clinton's press conference was that it called attention to the fact that life has been leaving signatures in its environment (be it earthly or extraterrestrial) for billions of years. In the years following the meteorite's discovery, it has become clear that to understand life's traces and-more importantly---effects on its environment, it is necessary to understand how life leaves its imprint and whether this can be distinguished from similar imprints left by abiotic processes. This is a central challenge in geobiology.", "isbn": "9781118280812", "publisher": "Wiley-Blackwell", "place_of_publication": "Hoboken, NJ", "publication_date": "2012", "pages": "228-249" }, { "id": "authors:nqyg6-dka08", "collection": "authors", "collection_id": "nqyg6-dka08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110518-083725900", "type": "book_section", "title": "Identification of Diazotrophic Microorganisms in Marine Sediment via Fluorescence In Situ Hybridization Coupled to Nanoscale Secondary Ion Mass Spectrometry (FISH-NanoSIMS)", "book_title": "Research on Nitrification and Related Processes", "author": [ { "family_name": "Dekas", "given_name": "Anne E.", "orcid": "0000-0001-9548-8413", "clpid": "Dekas-A-E" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "contributor": [ { "family_name": "Klotz", "given_name": "Martin G.", "clpid": "Klotz-M-G" } ], "abstract": "Growing appreciation for the biogeochemical significance of uncultured microorganisms is changing the focus of environmental microbiology. Techniques designed to investigate microbial metabolism in situ are increasingly popular, from mRNA-targeted fluorescence in situ hybridization (FISH) to the \"-omics\" revolution, including metagenomics, transcriptomics, and proteomics. Recently, the coupling of FISH with nanometer-scale secondary ion mass spectrometry (NanoSIMS) has taken this movement in a new direction, allowing single-cell metabolic analysis of uncultured microbial phylogenic groups. The main advantage of FISH-NanoSIMS over previous noncultivation-based techniques to probe metabolism is its ability to directly link 16S rRNA phylogenetic identity to metabolic function. In the following chapter, we describe the procedures necessary to identify nitrogen-fixing microbes within marine sediment via FISH-NanoSIMS, using our work on nitrogen fixation by uncultured deep-sea methane-consuming archaea as a case study.", "doi": "10.1016/B978-0-12-381294-0.00012-2", "isbn": "9780123812940", "publisher": "Elsevier", "place_of_publication": "Amsterdam", "publication_date": "2011", "pages": "281-305" } ]