Classification of Microorganisms & Taxonomy
11 sub-topics · Pages 113–168
1. Introduction
Microbial taxonomy provides the vocabulary and framework for describing biodiversity. The binomial system (Genus species, e.g., Escherichia coli) and hierarchical classification allow scientists worldwide to communicate unambiguously about organisms. The modern species concept in microbiology relies on DNA–DNA hybridisation (>70% similarity) or average nucleotide identity (>95–96% ANI) rather than morphology alone.
✏️ Fill in the Blank
1. The hierarchical taxonomic ranks from broadest to most specific are: Domain, Kingdom, Phylum, Class, Order, Family, Genus, and _______.
Show Answer
Species🔘 Multiple Choice
1. MALDI-TOF mass spectrometry is now widely used in clinical microbiology for bacterial identification. It works by:
Show Answer
Correct: B) Analysing the unique protein mass fingerprint (especially ribosomal proteins) of a bacterial isolate2. MALDI-TOF mass spectrometry is used in clinical microbiology to rapidly:
Show Answer
Correct: B) Identify microorganisms from protein mass spectra💬 Open-Ended Questions
1. What is MLSA (Multi-Locus Sequence Analysis) and how does it improve on single-gene (16S rRNA) classification? Describe how it is performed and what its output tells us.
Hint / Guidance
MLSA sequences 6–10 conserved housekeeping genes (e.g., gyrB, rpoB, atpD, recA) individually, aligns each, and concatenates sequences for phylogenetic analysis. Advantages over single 16S: greater resolution at species/subspecies level; reduces impact of HGT on any single gene; provides more phylogenetic signal. Output: concatenated phylogenetic tree with bootstrap values; allelic profiles for each locus can be stored in MLST databases (e.g., PubMLST) for epidemiological tracking.2. The Three-Domain System
Carl Woese's revolutionary 16S rRNA comparative analysis in 1977 revealed that all life divides into three primary domains: Bacteria, Archaea, and Eukarya. Archaea, though structurally prokaryotic (no nucleus), share key molecular features with eukaryotes (histones, RNA polymerase structure, similar transcription/translation machinery), placing them on the eukaryote branch of the universal tree of life.
✏️ Fill in the Blank
1. Microorganisms sharing greater than _______ % 16S rRNA gene sequence similarity are generally considered members of the same species.
Show Answer
97🔘 Multiple Choice
1. The concept of 'horizontal gene transfer' (HGT) complicates phylogenetic classification of bacteria because:
Show Answer
Correct: B) Genes acquired from distantly related organisms can make a bacterium appear phylogenetically related to the donor, not its true ancestors💬 Open-Ended Questions
1. Two environmental isolates have 16S rRNA similarity of 98.5% but are found in very different habitats and have distinct physiological properties. How would you determine whether they are the same or different species?
Hint / Guidance
98.5% 16S similarity is above the 97% threshold but not definitive — whole-genome methods needed. (1) Calculate ANI: if ≥ 95%, same species; < 95%, separate species. (2) DNA-DNA hybridisation: ≥ 70% → same species. (3) Phenotypic characterisation: key distinguishing traits (temperature/pH range, substrate utilisation). (4) Ecological and physiological data support separate ecotypes even if genetically similar. Polyphasic approach: combine genomic + phenotypic + ecological data for final classification.2. Explain the limitations of using 16S rRNA gene sequencing alone for bacterial classification. What complementary approaches are now recommended?
Hint / Guidance
Limitations: conserved region; different species may share >97% identity (e.g., Bacillus cereus group); does not capture functional diversity; HGT can affect rRNA operons. Complementary approaches: whole-genome sequencing (Average Nucleotide Identity, ANI ≥95% = same species); MLST (multi-locus sequence typing); digital DNA-DNA hybridisation (dDDH ≥70%); MALDI-TOF proteomics; physiological/biochemical tests. Genome-based taxonomy now replaces DDH as gold standard.3. Prokaryotes
Bacteria represent Earth's most metabolically diverse group of organisms. They range from strict aerobes to obligate anaerobes, from mesophiles to hyperthermophiles, from free-living to obligate intracellular parasites. Their metabolic repertoire — photosynthesis, chemolithotrophy, fermentation, nitrogen fixation — drives the biogeochemical cycles on which all eukaryotic life depends.
✏️ Fill in the Blank
1. The numerical value used to define bacterial species boundaries by whole-genome comparison is called Average Nucleotide Identity, abbreviated as _______.
Show Answer
ANI🔘 Multiple Choice
1. Which of the following describes a 'strain' in bacterial taxonomy?
Show Answer
Correct: B) A group of organisms below the species level, sharing a common ancestor and specific distinguishing characteristic💬 Open-Ended Questions
1. Explain why Archaea were originally classified as bacteria (Archaebacteria) for nearly 200 years, and what specific evidence ultimately demonstrated that they form a completely separate domain.
Hint / Guidance
Morphological similarity (no nucleus, similar size) led to grouping as 'bacteria'. Carl Woese's 1977 16S rRNA analysis showed Archaea form a separate monophyletic clade. Key evidence: (1) rRNA sequences form distinct lineage closer to Eukarya; (2) Ether-linked lipids (archaeol) vs. ester-linked in bacteria; (3) No peptidoglycan — S-layer or pseudomurein instead; (4) RNA polymerase structure resembles eukaryotic Pol II; (5) Histones present (like eukaryotes); (6) Introns present in some archaeal genes.2. What is a 'type strain' and why is it important in microbial taxonomy?
Hint / Guidance
Type strain: original strain upon which a species description is based; deposited in public culture collections (ATCC, DSM, NCIMB); serves as reference point for that species name. Importance: ensures nomenclatural stability — when a species is reassigned, the type strain retains the original name; allows comparison of new isolates against a defined standard; required for formal species description publication in IJSEM.4. Microbial Eukarya
Eukaryotic microorganisms span four major groups: protozoa (heterotrophic, often parasitic), algae (photosynthetic, aquatic primary producers), fungi (saprotrophic decomposers and mutualists), and slime moulds (unusual life cycles). Their complex cell structure enables ecological roles impossible for prokaryotes, including phagocytic predation of bacteria and formation of macroscopic fruiting bodies.
✏️ Fill in the Blank
1. The international standard for naming bacteria is governed by the International Code of Nomenclature of _______ (ICNP).
Show Answer
Prokaryotes2. The hierarchical classification system from broadest to most specific ends with the rank of _______.
Show Answer
Species🔘 Multiple Choice
1. The Bergey's Manual of Systematic Bacteriology is significant in microbiology because it:
Show Answer
Correct: B) Is the authoritative reference for bacterial taxonomy, classification, and nomenclature💬 Open-Ended Questions
1. Describe the phage typing method and explain how it is used to track the source of a bacterial outbreak.
Hint / Guidance
Phage typing: different strains of a bacterial species have different surface receptors; a panel of bacteriophages each lyses only strains with specific receptors; susceptibility pattern = phage type. Application: during a Salmonella outbreak, isolates from patients, food samples, and animal sources are phage-typed; matching phage types link isolates to a common source (e.g., contaminated poultry batch); has been largely replaced by whole-genome sequencing (WGS) for higher resolution.5. Western Blot & Phage Typing
Western blot uses antibodies to identify specific proteins after SDS-PAGE separation, providing both size and identity information — the diagnostic gold standard for HIV confirmation and prion detection. Phage typing exploits the exquisite host specificity of bacteriophages: a strain's susceptibility pattern to a panel of phages serves as a molecular fingerprint, invaluable in epidemiological outbreak investigations.
✏️ Fill in the Blank
1. The approach to taxonomy that integrates phenotypic, genotypic, and phylogenetic information to classify organisms is called _______ taxonomy.
Show Answer
Polyphasic2. A phylogenetic tree based on molecular sequences is called a _______ tree.
Show Answer
Phylogenetic💬 Open-Ended Questions
1. Why is the 16S rRNA gene considered an ideal molecular marker for bacterial classification? Describe its properties that make it suitable for phylogenetic analysis.
Hint / Guidance
Universally present in all prokaryotes; contains both conserved regions (for universal primer design) and nine variable regions (V1–V9) for species/genus discrimination; evolves at a consistent, slow rate suitable for deep phylogenetic analysis; multiple copies per cell (high sensitivity); well-curated databases (SILVA, NCBI) for comparison.2. Compare natural classification with artificial classification in microbiology, giving examples of each.
Hint / Guidance
Artificial (phenetic): groups by overall similarity of observable characters regardless of evolutionary relationship; e.g., grouping all gram-positive cocci together (includes Staphylococcus and Streptococcus which are not closely related). Natural (phylogenetic): reflects evolutionary history; based on homologous characters and molecular data; e.g., placing Archaea as separate domain. Natural classification more informative for predicting properties and understanding evolution; artificial classification may still be useful clinically for rapid identification.6. Cladogram
Cladograms (phylogenetic trees) depict evolutionary relationships as branching diagrams based on shared derived characters. In microbiology, trees are constructed from molecular sequence data — primarily 16S/18S rRNA — rather than morphology, because convergent evolution frequently produces similar structures in unrelated lineages. Branch lengths represent evolutionary distance (sequence divergence).
✏️ Fill in the Blank
1. The technique that sequences all genetic material in an environmental sample without culturing is called _______.
Show Answer
Metagenomics🔘 Multiple Choice
1. Phylogenetic trees used in microbial taxonomy are most commonly constructed based on:
Show Answer
Correct: C) 16S rRNA gene sequences💬 Open-Ended Questions
1. Distinguish between genotypic and phenotypic methods of bacterial classification. Provide two examples of each and discuss the advantages and limitations of each approach.
Hint / Guidance
Genotypic: 16S rRNA sequencing (precise, culture-independent but cannot detect phenotypic differences), ANI/DDH (whole-genome, gold standard for species but expensive). Phenotypic: Gram staining (rapid, cheap but not phylogenetically informative), biochemical tests/Biolog plates (functional characterisation but convergent evolution causes misclassification). Best practice (polyphasic taxonomy): integrate both approaches.2. How has whole-genome sequencing (WGS) transformed bacterial species identification and outbreak investigation compared to traditional methods?
Hint / Guidance
Traditional: biochemical tests (API strips), 16S rRNA sequencing, serotyping — days/weeks; limited resolution. WGS advantages: single nucleotide polymorphism (SNP) analysis can distinguish outbreak strains at near-clonal level; ANI replaces DDH for species delineation; detects resistance genes (resistome) and virulence factors directly; core genome MLST tracks transmission chains; turnaround: 24–48 h. Used in food safety: WGS traced 2011 E. coli O104:H4 outbreak to fenugreek sprouts from a specific German farm.7. Complex Viruses
Complex viruses such as T4 bacteriophage are marvels of nanoscale engineering: an icosahedral head (DNA genome), contractile tail (sheath), baseplate, and tail fibres that recognise specific surface receptors on E. coli. The entire assembly process is genetically encoded and self-assembles in the infected cell — producing hundreds of progeny phages within 25 minutes.
🔘 Multiple Choice
1. Which of the following is a limitation of phenotypic classification in microbiology?
Show Answer
Correct: B) Distantly related bacteria can have identical morphologies (convergent evolution)2. The species concept most commonly used in microbiology defines a bacterial species as a group of strains sharing ≥___ % DNA-DNA hybridisation identity.
Show Answer
Correct: B) 70%💬 Open-Ended Questions
1. A microbiologist isolates a novel bacterium from deep-sea sediment. Outline the steps she would take to classify and formally name this organism according to current microbiological standards.
Hint / Guidance
(1) Pure culture isolation; (2) 16S rRNA sequencing and BLAST comparison — if <97% similarity to known species, likely novel; (3) Phylogenetic tree construction; (4) ANI/DDH with closest relatives; (5) Phenotypic characterisation (morphology, Gram stain, growth conditions, biochemistry); (6) Formal description in IJSEM; (7) Type strain deposited in two international culture collections; (8) Binomial name proposed following ICNP rules.2. What are the main phenotypic characteristics used to identify bacteria in a clinical laboratory, and what are the limitations of phenotypic identification?
Hint / Guidance
Phenotypic: Gram stain (cell wall); cell morphology (cocci/bacilli/spirilla); spore formation; motility; colony morphology; biochemical tests (catalase, oxidase, sugar fermentation, API strip); oxygen requirements; growth temperature range. Limitations: many tests require 24–72 h; some phenotypically identical strains differ genotypically; biochemical profiles can vary with growth conditions; cannot identify non-culturable organisms; emerging drug-resistant variants may have altered phenotypes. Modern labs increasingly use MALDI-TOF + WGS for definitive ID.8. Growing Viruses
Since viruses are obligate intracellular parasites, they cannot be cultured on agar plates. Instead they are propagated in cell culture lines (Vero cells, HeLa cells), in embryonated hen eggs, or in whole animal models. Viral titres are determined by plaque assay — each plaque (clear zone in a cell monolayer) represents infection initiated by a single infectious virion.
🔘 Multiple Choice
1. The DNA-DNA hybridisation (DDH) threshold of 70% similarity has traditionally been used to define a bacterial species. This method is now being replaced by:
Show Answer
Correct: B) Average Nucleotide Identity (ANI) ≥ 95% threshold from whole-genome sequencing2. In the binomial nomenclature system, which part of the name is always written in lowercase?
Show Answer
Correct: C) Species epithet💬 Open-Ended Questions
1. Explain the concept of 'candidatus' status in bacterial taxonomy. When is this designation used, and what is required for a microorganism to be formally named?
Hint / Guidance
'Candidatus' is used for well-characterised but uncultured microorganisms (e.g., 'Candidatus Pelagibacter ubique'). Used when organism has been detected by molecular methods (16S rRNA, single-cell genomics, metagenomics) but cannot be grown in pure culture. For formal naming: must be isolated in pure culture, deposited as type strain in two recognised culture collections, and formally described in the International Journal of Systematic and Evolutionary Microbiology.9. Specialized Transduction
Specialised transduction occurs when a lysogenic phage (e.g., bacteriophage λ) undergoes imprecise excision from the host chromosome, accidentally packaging adjacent host genes alongside phage DNA. Classic examples include λ-gal and λ-bio transducing phages. This mechanism is responsible for the spread of some virulence factors and metabolic genes between bacterial strains.
🔘 Multiple Choice
1. In binomial nomenclature, which of the following is written correctly?
Show Answer
Correct: C) Escherichia coli (genus capitalised, species lowercase, both italicised)2. Cladistics groups organisms based on:
Show Answer
Correct: B) Shared derived characters (synapomorphies) from a common ancestor💬 Open-Ended Questions
1. What is the 'species concept' in microbiology and why is it more difficult to apply to bacteria than to eukaryotes? Discuss at least two alternative species concepts that have been proposed.
Hint / Guidance
Biological species concept (Mayr) — cannot apply to bacteria because they reproduce asexually and exchange genes across 'species' boundaries via HGT. Alternatives: (1) Phylogenomic species concept — based on ANI ≥ 95% or DDH ≥ 70%; practical but arbitrary threshold. (2) Ecotype concept (Cohan) — bacteria of the same ecotype share ecological niche and undergo periodic selection sweeps; defines species by ecology rather than genetics. (3) Core genome concept — species defined by shared gene content. HGT makes any single concept problematic.10. Multiplication of DNA Virus
DNA viruses replicate their genomes using DNA-dependent DNA polymerases — either their own (large dsDNA viruses like herpesviruses, poxviruses) or the host cell's (small dsDNA viruses like papillomaviruses). The lytic cycle (attachment → penetration → biosynthesis → assembly → release) produces ~100–1000 progeny per cell and is inherently destructive to the host. The lysogenic cycle allows long-term coexistence of phage genome with host DNA.
🔘 Multiple Choice
1. A phylogenetic tree that shows the evolutionary relationships among organisms based on shared derived characters is called a:
Show Answer
Correct: B) Cladogram2. Which rRNA gene is most commonly used as a 'molecular clock' for bacterial phylogeny?
Show Answer
Correct: B) 16S rRNA💬 Open-Ended Questions
1. How does horizontal gene transfer affect our ability to construct accurate phylogenetic trees for bacteria? What strategies do microbiologists use to minimise this problem?
Hint / Guidance
HGT transfers genes between distantly related lineages, creating conflicts between gene trees and organismal trees (reticulate evolution). Strategy: (1) Use core genome phylogeny (genes universally conserved and rarely transferred, e.g., rRNA genes, ribosomal proteins); (2) Concatenate multiple conserved marker genes (MLSA — multi-locus sequence analysis); (3) Detect HGT events bioinformatically (unusual GC content, codon usage bias, synteny breaks); (4) Construct 'species trees' averaging across many loci.11. Pathways of Multiplication for RNA-Containing Viruses
RNA viruses face a unique challenge: host cells lack RNA-dependent RNA polymerase so each RNA virus must encode its own. Positive-sense RNA viruses (e.g., poliovirus) use genomic RNA directly as mRNA; negative-sense RNA viruses (influenza) must first transcribe their genome into mRNA. Retroviruses (HIV) reverse-transcribe RNA → DNA via reverse transcriptase, integrating permanently into the host chromosome as a provirus — explaining why HIV infection is lifelong.
🔘 Multiple Choice
1. Which property of the 16S rRNA gene makes it useful for distinguishing closely related species?
Show Answer
Correct: B) Its variable regions (V1–V9) that differ between species within otherwise conserved flanking sequences2. A polyphasic taxonomy approach combines:
Show Answer
Correct: B) Genotypic, phenotypic, and phylogenetic data💬 Open-Ended Questions
1. Describe the role of culture collections (such as ATCC or DSMZ) in microbial taxonomy and research. Why is depositing a type strain in two separate culture collections a requirement for valid species description?