Signature Sequences & Phylogenetic Probes

Signature Sequences

• Short oligonucleotides unique to specific groups of organisms
• Signature sequences define a domain
• Signature sequences can identify a specific group within a domain
• Signature sequences can identify a particular genus or species
• Signature sequences are useful for placing newly isolated organisms into their correct phylogenetic group
• Used in the design of specific nucleic acid probes
• A nucleic acid probe is a DNA or RNA oligonucleotide complementary to a sequence in a target gene or RNA
• Nucleic acid probes and their targets hybridize when they come together

Phylogenetic Probes

• Nucleic acid strands that can be labeled and used to hybridize to a complementary nucleic acid from a mixture
• Phylogenetic probes can be general or specific
• Phylogenetic probes can target different domains, such as Bacteria, Archaea, and Eukarya
• Phylogenetic probes can be designed to target groups within a domain like members of individual families, genera, and species

FISH (Fluorescent In Situ Hybridization)

• FISH is a method to see where probes bind to cellular ribosomes microscopically when a fluorescent dye is attached to the probe
• Cells are treated to allow penetration of the probe-dye mixture
• After hybridization, the fluorescent cells are viewed under a fluorescent microscope
• Can track organisms directly in the environment using FISH
• Is used to identify specific pathogens from patient specimens or environmental samples
• FISH obviates the need to grow micro-organisms in culture

Microbial Community Analysis

• Can be performed using PCR amplification and sequencing analysis
• From environmental sequences, a phylogenetic tree can be generated that shows the different rRNA genes present in the community
• Specific organisms can be inferred without culturing techniques
• Microbial community analysis is a major aspect of microbial ecology research today
• Environmental genomics/Metagenomics aids in understanding microbial community structure and microbial interactions

DGGE (Denaturing Gradient Gel Electrophoresis)

• DGGE separates genes of the same size that differ in their melting (denaturing) profile
• Differences that impact that melting profile occur due to differences in base sequence
• The denaturant is a mixture of urea and formamide
• Strands melt at different denaturant concentrations

T-RFLP (Terminal Restriction Fragment Length Polymorphism)

• A target gene (rRNA) is amplified by PCR
• One of the primers used in PCR is labeled with a fluorescent dye
• PCR products are digested using restriction enzymes
• Produces a series of fragments; the quantity reflecting the number of restriction sites present
• The fragments are run on a gel
• The resulting pattern reflects the rRNA sequence variation in the community

Environmental Genomics (Metagenomics)

• DNA is cloned from a microbial community and sequenced
• Detects as many genes as possible
• Provides a picture of the gene pool in the environment
• Can detect genes that are not amplified by current PCR primers
• A tool for assessing the phylogenetic and metabolic diversity of an environment

Ribotyping

• Ribotyping does not involve sequencing
• Ribotyping reports the specific pattern of bands (DNA fingerprint)
• DNA from an organism is digested with restriction enzymes, and fragments separated by gel electrophoresis
• Fragments are transferred to nylon membrane and probed with SSU rRNA gene probes
• Differences in the 16S rRNA sequence translate into the presence or absence of cut sites
• The DNA pattern is compared with reference patterns in a database
• The DNA banding pattern (ribotype) is unique and diagnostic

Identification of Bacteria in Laboratory Culture

• Success in culturing a micro-organism depends on:
  • Understanding its nutritional requirements
  • Supplying appropriate levels of nutrients in culture
  • Nutrient proportions need to be supplied correctly in a culture medium
• Macronutrients include:
  • Carbon and Nitrogen; required by all cells
  • Phosphorus; is important for the Synth of nucleic acids and phospholipids
  • Sulfur; for a structural role in some amino acids and vitamins
  • Potassium; required by all organisms, including for Enzymes
  • Calcium; to stabilize cell walls
  • Magnesium; to stabilize ribosomes, nucleic acids, and enzymes
  • Sodium; required by some, but not all, reflecting habitat
  • Iron; plays a major role in cellular respiration
• Some micro-organisms produce iron-binding siderophores

Culture Media

• Solutions used to grow micro-organisms in the laboratory
• Careful attention must be paid to the selection and preparation of media for a successful culture
• Two main classes of culture media: defined and complex
• Defined media are prepared with precise amounts of highly purified inorganic or organic chemicals added to distilled water
• The exact chemical composition with a defined medium is known
• Of major importance is the carbon source
• The nature and concentration of said carbon source depends on the organism to be cultured
• Complex media is used in cases where knowing media composition is not critical
• Complex media may contain animal or plant products
  • Casien (milk protein)
  • Beef (beef extract)
  • Soybeans (tryptic soy broth)
  • Yeast cells (yeast extract)
• A selective medium contains compounds that selectively inhibit the growth of some micro-organisms but not others
• A differential medium contains an indicator (typically a dye) and allows the ability to differentiate particular chemical reactions during growth

Laboratory culture of Microorganism

• Once a culture media has been selected and made sterile
• Organisms can be inoculated and the culture incubated to support growth
• In the lab, a pure culture will be inoculated

Culture-Dependent Analysis of Microbial Communities

• Can be applicable to biotechnology
• Has uses in industrial and environmental microbiology
• Involves enrichment: separation of different organisms from a microbial community
• Involves obtaining pure cultures
• Enrichment culture establishes selective conditions favoring a desired organism and counter-selective ones against undesired micro-organisms
• Duplicates the resources and conditions of a niche environment
• Enrichment cultures are established placing inoculum (obtained from natural habitat) in in a selective media and incubated under specific conditions
• Matinus Beijerinck first isolated the N₂ -fixing bacterium Azotobacter using the classic enrichment strategy

The Winogradsky Column

• The Winogradsky Column is an artificial microbial ecosystem and a long-term source of bacteria for enrichment cultures
• Winogradsky columns have been routinely used to isolate:
  • Purple and green phototrophic bacteria
  • Sulfate-reducing bacteria
  • Many other anaerobes
• Within Winogradsky columns:
  • Algae and cyanobacteria appear at the top (oxic)
  • Decomposition in the mud led to the formation of organic acids, alcohols, and H₂
  • All providing a suitable environment for sulfate-reducing bacteria
  • Sulfide from the sulfate reducers lead to the development of purple and green sulfur bacteria (anoxygenic phototrophs)
• Winogradsky columns can be used to enrich both aerobes and anaerobes