Hydrocarbon and Lipid Microbiology Protocols

1. Front Matter

   Pages i-x

   PDF

2. Introduction to Single-Cell and Single-Molecule Methods

   • Wei E. Huang

   Pages 1-3

3. Single-Cell Cultivation in Microfluidic Devices

   • Sattar Taheri-Araghi, Suckjoon Jun

   Pages 5-16

4. Single-Cell Bacterial Bioreporter Assays to Measure Hydrocarbons

   • Robin Tecon

   Pages 17-28

5. Whole Genome Amplification of Single Bacterial Cells

   • Paul Scott, Alan W. Walker

   Pages 29-41

6. Detection of Specific mRNA Molecules in Hydrocarbon-Degrading Bacteria

   • Juhyun Kim, Víctor de Lorenzo

   Pages 43-50

7. Localization of Proteins Within Intact Bacterial Cells Using Fluorescent Protein Fusions

   • Veronica Wells Rowlett, William Margolin

   Pages 51-63

8. Single Cell Microbial Ecophysiology with Raman-FISH

   • Daniel Read, Wei E. Huang, Andrew S. Whiteley

   Pages 65-76

9. Single-Cell Metabolomics

   • Yun Wang, Yizhi Song, Ian P. Thompson, Jian Xu, Wei E. Huang

   Pages 77-90

10. Marrying Single Molecules to Single Cells: Protocols for the Study of the Bacterial Proto-ring Components Essential for Division

    • A. Sánchez-Gorostiaga, A. I. Rico, P. Natale, M. Krupka, M. Vicente

    Pages 91-118

11. Single-Cell Biotechnology for Uncultured Microorganisms

    • Peiran Zhang, Yizhi Song, Ian P. Thompson, Bo Ma, Jian Xu, Wei E. Huang

    Pages 119-131

12. Single-Cell and Systems Biology Tools for Biofuel Production

    • Tingting Wang, Yuetong Ji, Yun Wang, Yuehui He, Jing Jia, Qiang Hu et al.

    Pages 133-144

13. Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT)

    • Roland Hatzenpichler, Victoria J. Orphan

    Pages 145-157

14. Flow Cytometric Determination of Microbial Abundances and Its Use to Obtain Indices of Community Structure and Relative Activity

    • Josep M. Gasol, Xosé Anxelu G. Morán

    Pages 159-187

15. Protocols for Visualizing Horizontal Gene Transfer in Gram-Negative Bacteria Through Natural Competence

    • Melanie Blokesch

    Pages 189-204

16. Correction to: Marrying Single Molecules to Single Cells: Protocols for the Study of the Bacterial Proto-ring Components Essential for Division

    • A. Sánchez-Gorostiaga, A. I. Rico, P. Natale, M. Krupka, M. Vicente

    Pages C1-C1

This Volume presents relevant single-cell and single-molecule approaches in the study of microbes producing and utilizing hydrocarbons and lipids. While generically applicable for all microorganisms, the approaches described are, wherever possible, adapted to the field of study of hydrocarbon and lipid microbiology. The methods include basic procedures for isolating single cells by means of microfluidics and flow cytometry, and their cultivation in arrays as pure clones; for isolating, amplifying and sequencing single-cell genomes and transcriptomes; and for analysing single-cell metabolomes by means of Raman spectroscopy. Single-molecule approaches include the use of protein:fluorescent dye fusions for protein localization and methods for the production of cell division protostructures and lipid monolayers. Methods for the functional analysis of single cells include detection of metabolically active (protein-synthesizing) cells in environmental samples by bioorthogonal non-canonical amino acid tagging, Raman spectroscopy combined with stable isotope labelling and fluorescent in situ hybridisation, and visualization of single cells participating in gene transfer activity. Lastly, protocols are presented for single-cell biotechnological applications, including biofuel production.
Hydrocarbon and Lipid Microbiology Protocols
There are tens of thousands of structurally different hydrocarbons, hydrocarbon derivatives and lipids, and a wide array of these molecules are required for cells to function. The global hydrocarbon cycle, which is largely driven by microorganisms, has a major impact on our environment and climate. Microbes are responsible for cleaning up the environmental pollution caused by the exploitation of hydrocarbon reservoirs and will also be pivotal in reducing our reliance on fossil fuels by providing biofuels, plastics and industrial chemicals. Gaining an understanding of the relevant functions of the wide range of microbes that produce, consume and modify hydrocarbons and related compounds will be key to responding to these challenges. This comprehensive collection of current and emerging protocols will facilitate acquisition of this understanding and exploitation of useful activities of such microbes.

• Bioreporters
• Functional flow cytometry
• Metabolomics
• Protein localisation with GFP fusions
• Proteomics
• Single-cell genomics
• Single-molecule science
• biochemical engineering

• School of Biological Sciences, University of Essex, Colchester, UK

  Terry J. McGenity

• Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

  Kenneth N. Timmis

• Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

  Balbina Nogales

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