Microbiology (MICR2211)

  • Topics covered:
    • Microbial evolution and systematics
    • Earliest Life Forms
    • Molecular Microbial Phylogeny
    • Identification of bacteria in laboratory cultures and in nature

The Microbial World

  • Classification of living organisms:
    • Organisms are classified into three major groups called domains.
    • Properties shared among members of each domain distinguish them from members of other domains.
    • Many properties can be shared among different domains, indicating a possible common ancestor.

Molecular Phylogeny

  • Definition:
    • Phylogeny refers to the evolutionary history of a group of organisms.
  • Inference:
    • Phylogeny is derived indirectly from nucleotide sequence data.
    • The premise is that bacteria (and all organisms) are related by descent, with the sequence of a bacterium’s genome recording its ancestral lineage.

Evolutionary Analysis

  • Definition:
    • Evolution is characterized as a process of inherited nucleotide sequence change.
  • Analysis:
    • Analyzing DNA sequence differences among bacteria helps reconstruct their phylogenetic history.

Genes Used in Phylogenetic Analysis

  • Key genes:
    • 16S rRNA
    • 18S rRNA (eukaryotes)
  • Classification:
    • Both are classified as small subunit ribosomal RNA (SSU rRNA).

Importance of SSU rRNA in Phylogenetic Analysis

  • Advantages of SSU rRNA genes:
    • Universally distributed across various organisms.
    • Functionally constant.
    • Sufficiently conserved, meaning they change slowly over time.
    • Adequate length that provides a good overview of evolutionary relationships.

Contributions of Carl Woese

  • Established the three domains of life using SSU rRNA.
  • Recognition:
    • Awarded the 2003 Craford Prize for his work.
  • Ribosomal Database Project II contains over 440,000 sequences of SSU rRNA.

Additional Genes for Phylogenetic Analysis

  • 23S large-subunit rRNA (LSU rRNA):
    • Highly informative for phylogenetic studies.
  • Other relevant genes:
    • Protein synthesis elongation factor Tu (Ef-Tu)
    • Heat shock protein Hsp60
    • tRNA synthetases

Limitations of SSU rRNA

  • Variation:
    • The level of variation in SSU rRNA gene sequences may not be sufficient for distinguishing closely related bacterial strains.
  • Single gene drawback:
    • Relying on one gene (e.g., recA) can be misleading as it may not be universally present in all microorganisms.
    • It can be rectified by utilizing multiple genes for analysis.

Steps in Evolutionary Analysis

  • Methodology:
    1. Obtaining DNA sequences
    2. Performing sequence alignment
    3. Constructing phylogenetic trees
    4. Tree reconstruction

Molecular Phylogeny and Sequence Analysis

  • Sequence alignment:
    • Initial step involves aligning sequences of interest with homologous (orthologous) genes from other strains or species.
  • Phylogenetic tree characteristics:
    • A graphic representation illustrating relationships among sequences.
    • Comprised of nodes (representing organisms) and branches (showing relationships).
    • Branch lengths indicate the number of changes that occurred along that branch.

Tree Structure

  • Node nuances:
    • Rotating around a node does not alter the tree topology.
    • Example: the positions of lineages can be rotated without changing branching patterns.

Universal Phylogenetic Tree

  • Construction:
    • Determined through the comparative analysis of rRNA gene sequences.
    • Involved analyzing over 30 genes from 190 species spanning Bacteria, Archaea, and Eukarya.

Learning Outcomes

  • Key topics discussed include:
    • 12.8 The Species Concept in Microbiology
    • 12.9 Taxonomic Methods in Systematics
    • 12.10 Classification and Nomenclature
  • Reference material:
    • Brock Biology of Microorganisms, 14th edition, pages 393-399.

The Species Concept in Microbiology

  • Biological species concept:
    • Not applicable to prokaryotes due to their haploid nature and lack of sexual reproduction.
  • Alternative concept:
    • Phylogenetic species concept defines a prokaryotic species as a group of strains, closely clustering based on DNA sequences of multiple genes and distinct from other groups.

Current Definition of Prokaryotic Species

  • Definition details:
    • A prokaryotic species is characterized by:
    • High degree of similarity in several independent traits among strains.
    • ≥70% DNA–DNA hybridization.
    • ≥97% 16S rRNA gene sequence identity.

16S rRNA in Taxonomy

  • Role in species identification:
    • 16S rRNA gene sequences are regarded as the "gold standard" for new species identification and description.
  • Specification:
    • A new species is proposed if its 16S rRNA gene sequence differs by more than 3% from any known strain, and a new genus if the difference exceeds 5%.

Microbial Systematics

  • Definition:
    • Systematics is the study of the diversity of organisms and their relationships, linking phylogeny with taxonomy.
  • Characterization and naming:
    • Organisms are characterized, named, and grouped based on natural relationships.

Traditional vs. Modern Taxonomy

  • Traditional methods:
    • Relied on phenotypic characteristics.
  • Modern incorporation:
    • Now includes DNA sequences along with phenotypic data (polyphasic approach).
    • Genotypic (genome-level)
    • Phenotypic (traits and characteristics)
    • Phylogenetic (evolutionary relationships)
  • Importance of habitat and ecology in classification.

Phenotypic Analysis

  • FAME (Fatty Acid Methyl Ester) Analysis:
    • Identifies the types and proportions of fatty acids present in the cytoplasmic and outer membranes.
    • Important phenotypic trait used in clinical, public health, and food inspections, beneficial for characterizing new species.

Drawbacks of FAME Analysis

  • Standardization challenges:
    • Requires rigid standardization since fatty acid profiles change with temperature, growth phase, and medium.
  • Growth conditions limitation:
    • Limited to organisms that can grow under conditions matching those in established databases.

Genotypic Analysis

  • DNA-DNA hybridization:
    • If two organisms share numerous identical genes, their DNA is expected to hybridize in proportion to similarity of gene sequences.
    • It provides a rough similarity index to each other, complementing 16S rRNA sequencing.

DNA Profiling Methods

  • Repetitive extragenic palindromic PCR (rep-PCR):
    • Based on highly conserved repetitive DNA elements scattered throughout the bacterial genome.
    • Variability in number and position among strains leads to strain-specific banding patterns when amplified.
  • Multilocus sequence typing (MLST):
    • Involves sequencing several different housekeeping genes from an organism and comparing them across strains.
    • Sensitive method, limited to strain differentiation.

Process of MLST

  • Steps:
    1. Isolate DNA from the sample.
    2. Amplify 6-7 target housekeeping genes.
    3. Sequence the amplified genes.
    4. Analyze alleles and compare with other strains to construct phylogenetic trees.

Definition of Prokaryotic Species

  • Operational definition:
    • A collection of strains sharing significant similarity in various traits.
    • Defined by:
    • DNA-DNA hybridization of 70% and greater.
    • 16S rRNA gene sequence identity of 97% or greater.

Classification and Nomenclature

  • Classification:
    • Organizing organisms into increasingly inclusive groups based on phenotypic similarity or evolutionary relationships.
    • Structure:
    • Species comprised of multiple strains.
    • Similar species grouped into genera.
    • Similar genera grouped into families.
    • Orders grouped into classes.
    • Classes grouped into phyla.
    • Phyla classified under domains.

Nomenclature in Microbiology

  • Definition:
    • Actual naming of organisms, applying formal rules.
    • Adherence to the binomial system of nomenclature for prokaryotes, resulting in genus and species names (e.g., Bacillus subtilis, Bacillus cereus, Bacillus megaterium).

Oversight of Taxonomy

  • International Committee on Systematics of Prokaryotes (ICSP):
    • Responsible for the oversight of nomenclature and taxonomy for Bacteria and Archaea.
  • The Bacteriological Code:
    • Governs the assignment of names for species and higher groups within Bacteria and Archaea.

Classification Limitations

  • Taxonomy as scientific judgment:
    • There is no official taxonomy for Bacteria and Archaea due to the subjective nature of classification.
    • The Taxonomic Outline of the Prokaryotes in Bergey’s Manual of Systematic Bacteriology is the most widely accepted classification framework.

Formal Recognition of New Species

  • Requirements for new species:
    • Deposition of the organism sample in two culture collections.
    • Official publication of the new species name and description in the International Journal of Systematic and Evolutionary Microbiology (IJSEM).

Culture Collections

  • Purpose:
    • Store viable cultures of microorganisms (frozen or freeze-dried) and act as repositories for type strains with taxonomic significance.
  • When describing a new species, one strain is designated as the nomenclatural type for future taxonomic comparisons.