6a and 6b

• Microorganisms


  Microbes that drive the biogeochemical cycles and deliver ecosystem services
• We know less than 1% of the microbial species on Earth!
• Tools to identify and quantify microorganisms

  • Microscopy
  • Culturing
  • DNA-based methods (genomics)

  
• Microscopy


  • Used to characterise phenotypes of individual strains/cultures, or must be combined with DNA-based methods in mixed microbial communities (i.e., "FISH")

  
• Culturing


  • Sometimes useful for assessing function/metabolism, also can be combined with DNA-based methods (i.e. "enrichments")
• DNA-based methods (genomics)


  • Most powerful method for characterising diversity of ALL microbes in a sample

  
• Culture-independent approach


  Allows direct identification of microbes in a sample without the need for cultivation; includes DNA-based or molecular methods

  
• Taxonomic marker gene


  Gene that is conserved among all taxa of interest; usually "housekeeping" genes (16S rRNA gene)
• Functional gene


  Gene that encodes a specific cellular or metabolic function; may not be conserved across all microbes in a community/sample
• 16S rRNA


  A sequence of DNA that encodes the RNA component of the smaller subunit of the bacterial ribosome

  
• Amplification of 16S rRNA genes by Polymerase Chain Reaction (PCR)

  Chain Termination (Sanger) Sequencing

  
• DNA sequencing


  Determining the sequences of nucleotides

  
• Culture-independent methods allow us to study the taxonomy, function and abundance of microorganisms in the environment

  
• 16S rRNA gene databases

  • The Ribosomal Database Project (RDP-II)
  • Greengenes

  
• Advanced Sequencing Technologies

  • Amplicon sequencing
  • Whole Genome Amplification
  • Metagenomics
  • Metatranscriptomics
  • Metaproteomics

  
• Amplicon sequencing


  PCR-based technique, most popular is Illumina MiSeq, can sequence 16S rRNA gene or functional genes

  
• Whole Genome Amplification


  Shotgun sequencing, DNA is first shredded into small fragments, which are sequenced individually and then assembled

  
• Metagenomics


  Sequencing of the collective genomes of all microbes in an environmental sample

  
• Metatranscriptomics


  Sequencing of RNA to identify actively transcribed genes, shows microbial activity

  
• Metaproteomics


  Study of all proteins from cells, identification of functional gene products, shows microbial activity