Lecture 16

Relative Abundance Plot of Bacteria

  • Understanding Relative Abundance

    • Question: Is the relative abundance plot of bacteria an average for all people?
    • Relative abundance refers to:
    • The total number of reads rather than a specific individual.
    • Can represent one sample that may correspond to an individual, depending on experimental design.

  • Primer Binding and Species

    • Primers bind to constant regions of bacterial DNA.
    • Primers are generally the same across species, targeting conserved regions flanking hypervariable regions.
    • The hypervariable region can alter primer use, but generally, binding is consistent.

  • Operational Taxonomic Units (OTUs)

    • OTUs are used to group 16S sequences and are often a proxy for species.
    • Definition is computational, not based on 16S rRNA alone.
    • Question: Could some microbes be unsequenceable due to a lack of highly conserved regions?
    • Yes, certain archaea exhibit divergent 16S sequences where standard primers may not work effectively.

Species in Microbiology

  • Definition of Species
    • No clear “biological species concept” in microbiology due to asexual reproduction and rapid acquisition of genetic material.
    • Species are delineated by sequence similarity, often a threshold of 97% similarity in the 16S rRNA gene.
    • Example:
    • E. coli K12 vs. E. coli O157:
      • 2.8% dissimilarity in their 16S rRNA.
      • Fostered evolutionary divergence estimated at ~100-140 million years ago; 4.5 million years ago they exhibited >99% similarity at 16S, yet 20-30% of the genome was different.

Limitations of 16S rRNA

  • 16S rRNA Molecule Limitations
    • Not ideal for delineating evolutionary relationships within microbial species.
    • Alternatives:
    • Multilocus Sequence Analysis (MLSA)
      • Protein-coding genes accumulate mutations faster than rRNA, allowing for better specification of species.
      • Uses conserved genes like recA and gyrB, which seldom undergo horizontal gene transfer.
      • Can analyze between 4 and over 1000 genes.

16S rRNA Amplicon Sequencing vs. Shotgun Metagenomic Sequencing

  • Choosing sequencing methods for microbiome studies:
    • Questions to Consider:
    • Why choose 16S rRNA sequencing over shotgun metagenomic sequencing?
    • What are the pitfalls of metagenomic sequencing?
  • Trade-offs:
    • Cost:
    • 16S library sequencing costs around $30 per sample.
    • Metagenomics can range in the hundreds of dollars per sample; this affects the number of samples that can be analyzed.
    • Analysis Complexity:
    • Metagenomic data analysis is complicated, time-consuming, and computationally intensive.
    • Insight Gained:
    • Can offer insights into species composition of microbial communities even with the simpler methods.

Diversity in Microbial Communities

  • Alpha Diversity

    • Definition: Measure of how many different taxa are present and how evenly they are distributed in a sample.
    • Question Examples:
    • Does gut microbiome diversity vary among individuals?
    • Are diet or disease driving these variations?

  • Beta Diversity

    • Definition: Quantifies how different the microbial communities are from one another across samples.

Lecture on Evolution

Organelles and Evolution

  • Role of Symbiosis in Evolution
    • Endosymbiosis as a key process in the evolution of organelles in eukaryotes, specifically mitochondria and chloroplasts.

Timeline of Evolutionary Events

  • Earth’s History:
    • Formation of the moon and early Earth (~4.5 billion years ago).
    • First prokaryotic life (~3.8 billion years ago).
    • Emergence of first eukaryotes (~2.1 billion years ago).

Evidence for Endosymbiotic Theory

  • Key evidential points for the endosymbiotic origin of mitochondria and chloroplasts:

    • Size and Replication:
    • Mitochondria and chloroplasts are similar in size to bacteria and replicate independently by binary fission.
    • Genomes:
    • They possess circular DNA genomes resembling bacterial genomes.
    • Ribosomes:
    • Contain bacterial-type (70S) ribosomes, unlike eukaryotic 80S ribosomes.
    • Phylogenetic Evidence:
    • rRNA and many genes are closely related to respective bacterial groups (α-proteobacteria for mitochondria, cyanobacteria for chloroplasts).
    • Antibiotic Sensitivity:
    • Similar antibiotic sensitivity to bacterial translation, but not to eukaryotic cytoplasm.
    • Membranes:
    • Both organelles have double membranes, indicative of an engulfment process.
    • Gene Transfer:
    • Many genes from organelles have migrated to the nuclear genome, indicating historical integration and dependency.

  • Mention of Lynn Margulis and her contributions to the understanding of endosymbiosis.

Lokiarchaeota and Eukaryotic Origin

  • Discovery and Significance:
    • Lokiarchaeota found in deep-sea sediments, with relatives existing in anaerobic environments.
    • Genomic content provides evidence that eukaryotes likely descended from this lineage due to presence of genes unique to eukaryotes.

Hypotheses on Eukaryotic Evolution

  • Serial Endosymbiosis Hypothesis:
    • Suggests a host cell capable of phagocytosis existed before acquiring a bacterium which became a mitochondrion.
  • Symbiogenesis Hypothesis:
    • Proposes that the acquisition of the mitochondrion spurred eukaryotic complexity.

Unique Organelles: Nitroplast

  • A nitroplast, a nitrogen-fixing organelle found in some algae, notably in Braarudosphaera bigelowii.
    • Originated from cyanobacterial endosymbionts that integrated into the host.

History of Eukaryotes and Multicellularity

  • Eukaryotes started to experiment with multicellularity approximately 541 million years ago during the Cambrian explosion.

Evolutionary Study through Experimental Evolution

  • Experimental Evolution Overview:

    • Defined as the study of evolution in controlled laboratory settings.
    • Key objectives:
    • Examine dynamics of evolution and rate of change.
    • Investigate repeatability of evolution and the interplay between phenotype and genotype changes.

  • Long-Term Evolution Experiment (LTEE):

    • Initiated by Richard Lenski in 1988, culturing E. coli populations in a minimal glucose medium enriched with citrate.
    • Conditions allowed for observation of evolutionary processes across over 73,500 generations.

Mutations and Adaptation in E. coli

  • Citrate Utilization in E. coli:

    • Wild-type E. coli does not utilize citrate under aerobic conditions due to metabolic inefficiency.

  • Genetic Changes:

    • Cit+ clones exhibited mutations where a 3000 base pair segment was duplicated, altering citrate transporter function in the presence of oxygen.

  • Mechanistic Insights:

    • Initial background mutations created suitable conditions for the Cit+ mutation to confer a fitness advantage, permitting refinement in metabolic pathway utilization.
Evolutionary Concepts:
  • Potentiation: Evolution of a genetic background that makes a trait accessible.
  • Actualization: Occurrence of a mutation that brings a trait into expression.
  • Refinement: Subsequent mutations improve expressed traits.

Loss of Function in Evolution

  • Case study of phototrophic purple bacteria in dark environments where loss of function mutations occurred, reflecting natural selection favoring efficiency when certain capabilities became redundant.