Microbial Taxonomy and Diversity: Bacteria, Archaea, Fungi, Protozoa, Viruses and Molecular Phylogeny
2. Broad classification of Bacteria and Archaea
Older schemes grouped bacteria by shape (cocci, bacilli, vibrios, spirilla, spirochaetes), Gram reaction, oxygen relations and spore formation; current classification is phylogenetic. Several phylum names were formally revised in 2021, so both forms appear in books.
| Phylum (older name) | Hallmarks | Examples |
|---|---|---|
| Pseudomonadota (Proteobacteria) | Largest group; Gram-negative; metabolically diverse; traditionally classes alpha to epsilon | Rhizobium, Rickettsia, Neisseria, Nitrosomonas, Escherichia, Pseudomonas, Vibrio |
| Bacillota (Firmicutes) | Gram-positive, low G+C; some form endospores | Bacillus, Clostridium, Staphylococcus, Streptococcus, Lactobacillus, Listeria |
| Actinomycetota (Actinobacteria) | Gram-positive, high G+C; many filamentous | Streptomyces (most natural antibiotics), Mycobacterium (mycolic acids), Corynebacterium |
| Cyanobacteria | Oxygenic photosynthesis with chlorophyll a and phycobilisomes; heterocysts fix N₂; ancestors of chloroplasts | Anabaena, Nostoc, Synechococcus |
| Bacteroidota, Chlamydiota, Spirochaetota | Gut anaerobes and gliders; obligate intracellular parasites with elementary and reticulate bodies; helical cells with periplasmic flagella | Bacteroides; Chlamydia; Treponema, Borrelia, Leptospira |
Archaea are not only extremophiles; they are abundant in oceans and soils, and no archaeal pathogen of humans is known. Methanogens (Methanobacterium, Methanosarcina) are strict anaerobes that make methane using unique coenzymes such as coenzyme M and F₄₂₀. Extreme halophiles (Halobacterium) need high NaCl, balance it with internal K⁺, and use bacteriorhodopsin, a retinal-containing light-driven proton pump. Hyperthermophiles such as Sulfolobus (hot acid springs, often sulphur-oxidising) and Pyrolobus (growing above 100 °C) belong to the Crenarchaeota (Thermoproteota); Thermoplasma lacks a wall. Ammonia-oxidising archaea (Nitrosopumilus) drive nitrification in the sea, the tiny Nanoarchaeum lives attached to another archaeon, and Asgard archaea are the closest known prokaryotic relatives of eukaryotes.
3. Eukaryotic microbes: yeasts, moulds and protozoa
Fungi are absorptive chemoorganoheterotrophs with walls of chitin and glucans and ergosterol in their membranes, the target of polyene and azole drugs. Yeasts are unicellular: Saccharomyces cerevisiae buds, Schizosaccharomyces pombe divides by fission, Candida albicans forms buds, pseudohyphae and true hyphae, and Cryptococcus neoformans is a capsulated basidiomycete yeast. Moulds grow as branched hyphae forming a mycelium; hyphae are septate (cross-walled) or coenocytic (aseptate, multinucleate). Dimorphic fungi such as Histoplasma capsulatum grow as moulds at 25 °C and as yeasts at 37 °C in tissue. Asexual spores are sporangiospores (inside a sporangium) or conidia (borne free on conidiophores); the kind of sexual spore defines the major groups. Fungi with no known sexual stage were once lumped as Deuteromycetes; DNA sequences, especially the ITS region, now place them.
| Group | Hyphae; sexual spore | Asexual spores; examples |
|---|---|---|
| Zygomycetes (Mucoromycota) | Coenocytic; thick-walled zygospore | Sporangiospores; Rhizopus, Mucor |
| Ascomycota | Septate; ascospores formed inside a sac, the ascus | Conidia; Aspergillus, Penicillium, Neurospora, Saccharomyces |
| Basidiomycota | Septate; basidiospores borne outside a club-shaped basidium | Mushrooms, rusts, smuts, Cryptococcus |
| Chytridiomycota | Simple, often unicellular | Flagellated zoospores, unique among fungi |
Protozoa are unicellular, mostly motile, heterotrophic eukaryotes, many forming resistant cysts that transmit infection. The classic grouping is by locomotion. Amoeboid forms move by pseudopodia (Entamoeba histolytica, amoebic dysentery). Flagellates include Giardia lamblia (no typical mitochondria), Trichomonas vaginalis, Trypanosoma brucei (sleeping sickness, by tsetse flies) and Leishmania donovani (kala-azar, by sandflies). Ciliates such as Paramecium carry cilia, a macronucleus and a micronucleus, and exchange nuclei by conjugation; Balantidium coli is the ciliate that infects humans. Apicomplexans (sporozoa) have an apical complex for invading host cells, no locomotory organelles in most stages and complex life cycles: Plasmodium, Toxoplasma, Cryptosporidium. Molecular phylogeny shows these four groups are not natural lineages; modern schemes place them in supergroups such as Excavata (Giardia, Trypanosoma), Amoebozoa (Entamoeba) and the SAR group (ciliates, apicomplexans).
4. Viruses and their classification
A virus is an acellular obligate intracellular parasite. Its particle, the virion, is a genome of DNA or RNA inside a protein capsid built of capsomeres, sometimes wrapped in a lipid envelope taken from a host membrane and studded with viral glycoproteins. Capsids are helical (tobacco mosaic virus), icosahedral (20 triangular faces and 12 vertices: adenovirus, poliovirus) or complex (the head-and-tail of phage T4, poxviruses). Most virions are 20–300 nm across. The ICTV (International Committee on Taxonomy of Viruses) assigns orders, families (-viridae), genera and species using genome type, capsid symmetry, envelope, host and sequence. The Baltimore scheme groups viruses by how they make mRNA, which is defined as positive (+) sense.
| Class and genome | Route to mRNA | Examples |
|---|---|---|
| I: dsDNA | Transcribed by a DNA-dependent RNA polymerase, host or viral | Herpesviruses, adenoviruses, poxviruses, phages T4 and λ |
| II: ssDNA | Converted to dsDNA, then transcribed | Parvoviruses, phage φX174 |
| III: dsRNA | Virion RNA-dependent RNA polymerase transcribes the genome | Rotavirus (segmented) |
| IV: (+)ssRNA | Genome acts directly as mRNA; naked RNA is infectious | Poliovirus, coronaviruses, hepatitis A virus, tobacco mosaic virus |
| V: (−)ssRNA | Virion RNA-dependent RNA polymerase copies the genome into mRNA | Influenza (segmented), rabies, measles, Ebola |
| VI: (+)ssRNA with reverse transcriptase | RNA → DNA by reverse transcriptase; DNA integrates | HIV and other retroviruses |
| VII: gapped dsDNA with reverse transcriptase | Transcribed to RNA, which is reverse-transcribed into new genomes | Hepatitis B virus, cauliflower mosaic virus |
5. Molecular approaches to taxonomy and phylogeny
Why rRNA genes? The 16S rRNA gene of prokaryotes (18S in eukaryotes) is present in every cell, does the same job everywhere, is rarely transferred horizontally, and is about 1,500 nucleotides long with a mosaic of conserved regions, where universal primers bind, and nine variable regions (V1–V9) that discriminate between taxa. It is excellent for placing an organism in a genus or family but too conserved to separate close species. By convention, strains below about 98.7% 16S identity belong to different species (an older cut-off of 97% defined operational taxonomic units), but identity above it does not prove they are the same species. Genome measures decide that: DNA–DNA hybridisation of 70% or more was the classic species criterion, and an average nucleotide identity (ANI) of about 95–96% corresponds to it. For fungi the ITS region is the standard barcode; MLSA of housekeeping genes (gyrB, rpoB, recA) resolves close relatives; MALDI-TOF mass spectra of ribosomal proteins identify isolates in clinical laboratories.
G+C content (mol%) ranges from roughly 25% to 75% among bacteria. Organisms differing by more than about 10% are unlikely to share a genus, but equal G+C does not show relatedness. It is found from the DNA melting temperature, since G·C pairs have three hydrogen bonds and stack more strongly; the Marmur–Doty relation in standard saline citrate is Tm = 69.3 + 0.41 (% G+C). Example: Tm = 89.8 °C gives (89.8 − 69.3)/0.41 = 50.0% G+C.
Trees. A phylogenetic tree joins taxa (tips) through nodes (inferred ancestors) by branches whose lengths show amount of change. Rooting with an outgroup gives direction in time; an unrooted tree shows relationships only. A clade (monophyletic group) is an ancestor and all its descendants. Trees are built by distance methods (UPGMA, which assumes a constant rate, and neighbour-joining), maximum parsimony, maximum likelihood or Bayesian inference, and bootstrap values give support for each branch. The number of possible bifurcating trees explodes: for n taxa there are (2n − 5)!! = 1 × 3 × 5 × … × (2n − 5) unrooted trees and (2n − 3)!! rooted ones, so 4 taxa give 3 unrooted and 15 rooted trees, and 5 taxa give 15 and 105.
Key takeaways
- Taxonomy is classification, nomenclature and identification; prokaryotic species are defined polyphasically around a type strain, and Woese's rRNA comparisons gave three domains: Bacteria, Archaea and Eukarya.
- Archaea have ether-linked isoprenoid lipids on glycerol-1-phosphate, no peptidoglycan, a eukaryote-like RNA polymerase and methionine initiators; they include methanogens, halophiles with bacteriorhodopsin, hyperthermophiles, marine ammonia oxidisers and the Asgard relatives of eukaryotes.
- Key bacterial phyla: Pseudomonadota (Gram-negative, diverse), Bacillota (low-G+C Gram-positives, endospores), Actinomycetota (high-G+C, Streptomyces, Mycobacterium), Cyanobacteria, Bacteroidota, Chlamydiota and Spirochaetota.
- Fungi have chitin walls and ergosterol; yeasts bud or divide, moulds form septate or coenocytic hyphae, and zygospores, ascospores and basidiospores define the major groups. Protozoa are grouped by locomotion into amoeboid, flagellate, ciliate and apicomplexan forms, which are not natural lineages.
- Viruses are classified by ICTV and by the seven Baltimore classes; (−)RNA and dsRNA virions carry an RNA-dependent RNA polymerase, (+)RNA is itself infectious, and classes VI and VII use reverse transcriptase. Viroids, satellites and prions are subviral.
- 16S rRNA places organisms (species cut-off about 98.7% by convention) but ANI of about 95–96% or DDH of 70% decides species; Tm = 69.3 + 0.41(% G+C); trees number (2n − 5)!! unrooted and (2n − 3)!! rooted, and Jukes–Cantor corrects p for multiple hits.
Practice questions (18)
Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.
The membrane lipids of archaea are best described as
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Answer: B — isoprenoid chains joined by ether bonds to glycerol-1-phosphate
Archaeal lipids are isoprenoid (phytanyl and related) chains ether-linked to glycerol-1-phosphate, the opposite stereochemistry to the glycerol-3-phosphate of bacteria and eukaryotes, which use ester-linked fatty acids, the tempting distractor. Sterols are eukaryotic, and mycolic acids belong to the mycobacterial wall.A new prokaryote is isolated from a hot spring at 85 °C. Which single observation would place it in the domain Archaea rather than Bacteria?
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Answer: D — Its membrane lipids are glycerol ethers of isoprenoid chains, not fatty-acid esters
Ether-linked isoprenoid lipids are found only in archaea, so they assign the domain. Hyperthermophily is the tempting distractor, but bacteria such as Aquifex and Thermotoga also grow above 80 °C. No nucleus with 70S ribosomes fits both prokaryotic domains, and Gram reaction and rod shape occur in both.Which of the following features do archaea share with eukaryotes but not with bacteria?
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Answer: A — Protein synthesis starts with methionine rather than formylmethionine; B — Promoter recognition by a TATA-binding protein, with an RNA polymerase closely resembling eukaryotic RNA polymerase II
Archaea and eukaryotes both initiate translation with unformylated methionine and transcribe with a complex, pol II-like RNA polymerase recruited by a TATA-binding protein (bacteria use sigma factors instead), part of why archaea are the closer relatives of eukaryotes. Peptidoglycan is a bacterial feature absent from both, and 70S cytoplasmic ribosomes are shared by bacteria and archaea, whereas eukaryotes have 80S.A filamentous, Gram-positive soil bacterium with DNA of high G+C content forms chains of aerial spores and produces streptomycin. It belongs to the phylum
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Answer: C — Actinomycetota (Actinobacteria), the high-G+C Gram-positives
This is Streptomyces: filamentous, high G+C, Gram-positive, sporulating, and the source of most natural antibiotics. Bacillota is the tempting distractor because it too is Gram-positive and spore-forming, but its members have low G+C and form endospores inside cells rather than chains of aerial spores. Cyanobacteria and Pseudomonadota are Gram-negative.Halobacterium salinarum makes ATP in the light without chlorophyll. The light-harvesting molecule responsible is
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Answer: A — bacteriorhodopsin, a retinal-containing protein that pumps protons out of the cell
Light isomerises the retinal of bacteriorhodopsin, which pumps H⁺ outward; the proton motive force drives ATP synthase. Halobacteria have no chlorophyll-based reaction centre, so bacteriochlorophyll (purple bacteria) and phycocyanin (cyanobacteria) are wrong; their red carotenoids protect against light but do not feed electrons to ATP synthase, which is driven by protons, not electrons.A septate mould forms its sexual spores inside a sac-like cell. It belongs to the
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Answer: B — Ascomycota
Ascospores form inside a sac, the ascus, the defining feature of Ascomycota (Aspergillus, Penicillium, Neurospora). Basidiomycota is the tempting distractor, being septate too, but its basidiospores are borne outside a club-shaped basidium. Zygomycetes are coenocytic and form zygospores, and chytrids have flagellated zoospores.Which pair of features distinguishes Rhizopus from Aspergillus?
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Answer: D — Rhizopus has coenocytic hyphae and sporangiospores; Aspergillus has septate hyphae and conidia
Rhizopus, a zygomycete, has aseptate multinucleate hyphae and makes sporangiospores inside sporangia (and zygospores sexually). Aspergillus, an ascomycete, has septate hyphae and bears conidia in chains on a conidiophore. The reversed pairing is the tempting distractor; neither forms basidiospores, and both are filamentous moulds.Which of the following protozoa are correctly described?
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Answer: A — Plasmodium: an apicomplexan with an apical complex, transmitted to humans by Anopheles mosquitoes; B — Giardia lamblia: a flagellate whose cysts in water transmit intestinal infection
Plasmodium is the classic apicomplexan, carried by Anopheles, and Giardia is a flagellate spread by cysts in contaminated water. Paramecium is a ciliate, not amoeboid, and it is the ciliates, not Entamoeba, that carry a macronucleus and a micronucleus; Entamoeba is amoeboid.A fungus isolated from a patient grows as a filamentous mould on agar at 25 °C but as budding yeast cells at 37 °C and in tissue. Which organism shows this behaviour?
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Answer: C — Histoplasma capsulatum, a thermally dimorphic fungus
Thermal dimorphism, mould in the environment and yeast at body temperature, is the hallmark of Histoplasma capsulatum and related pathogens. Saccharomyces is a yeast at all temperatures, while Rhizopus and Aspergillus remain filamentous moulds, Aspergillus growing as hyphae even in infected lung tissue.Why must a negative-sense single-stranded RNA virus carry an RNA-dependent RNA polymerase inside its virion?
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Answer: A — Its genome cannot be translated, and host cells have no enzyme to copy RNA into mRNA
A (−) genome is complementary to mRNA, so ribosomes cannot read it, and cells lack an RNA-dependent RNA polymerase to make (+) mRNA from it; the virus must bring its own. Reverse transcription and integration describe retroviruses (class VI), the tempting distractor. RNA polymerase II does not degrade RNA, and capsids are built from structural proteins, not the polymerase.Genomic nucleic acid, purified free of all viral proteins, is introduced into susceptible cultured cells. For which virus would this naked genome alone give rise to infectious progeny?
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Answer: D — Poliovirus, with a positive-sense single-stranded RNA genome
Poliovirus RNA is itself mRNA: ribosomes translate it into a polyprotein that includes the viral RNA-dependent RNA polymerase, so the full cycle follows. Influenza, measles and rotavirus genomes cannot be translated directly and need the polymerase packaged in the virion, so their protein-free RNA is not infectious. Segmentation is a distractor; the deciding property is the sense of the genome.Which of the following virus–Baltimore class pairings are correct?
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Answer: A — HIV: class VI, single-stranded RNA replicated through a DNA intermediate; B — Hepatitis B virus: class VII, gapped double-stranded DNA replicated through an RNA intermediate; D — Rotavirus: class III, segmented double-stranded RNA
HIV (a retrovirus) is class VI, hepatitis B virus is class VII and rotavirus is class III. Influenza virus has a negative-sense segmented RNA genome and belongs to class V, not IV.An infectious agent of potato plants consists only of a small circular single-stranded RNA of a few hundred nucleotides that encodes no protein and has no protein coat. It is a
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Answer: B — viroid, a naked non-coding RNA
A naked, non-coding, small circular RNA that infects plants (potato spindle tuber disease is the classic case) is a viroid. A satellite virus is the tempting distractor, but it encodes a capsid protein and needs a helper virus; prions contain no nucleic acid, and phages have protein capsids.The DNA of a bacterial isolate melts with Tm = 94.72 °C in standard saline citrate. Using Tm = 69.3 + 0.41 (% G+C), its G+C content in mol% is ____.
Numerical answer — type the value.
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Answer: 62
% G+C = (Tm − 69.3)/0.41 = (94.72 − 69.3)/0.41 = 25.42/0.41 = 62. A higher Tm means more G·C pairs, which have three hydrogen bonds and stronger stacking than A·T pairs.The number of distinct rooted, fully bifurcating trees for n taxa is (2n − 3)!! = 1 × 3 × 5 × … × (2n − 3). The number of rooted trees possible for 6 bacterial species is ____.
Numerical answer — type the value.
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Answer: 945
For n = 6, 2n − 3 = 9, so the count is 1 × 3 × 5 × 7 × 9 = 945. The unrooted count would be (2n − 5)!! = 1 × 3 × 5 × 7 = 105, the usual slip. The rapid growth is why tree-building programs search heuristically rather than testing every tree.Two aligned 16S rRNA gene sequences of 1,500 nucleotides differ at 120 positions (no gaps). Using the Jukes–Cantor correction d = −(3/4) ln(1 − 4p/3), where p is the proportion of differing sites, the evolutionary distance d in substitutions per site is ____ (to three decimal places).
Numerical answer — type the value.
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Answer: 0.085
p = 120/1500 = 0.080. 4p/3 = 0.10667, so 1 − 4p/3 = 0.89333 and ln(0.89333) = −0.11279. d = 0.75 × 0.11279 = 0.0846, which is 0.085 to three decimal places. The corrected distance exceeds p because some sites have changed more than once.Two soil isolates share 99.2% 16S rRNA gene identity, and their genomes give an average nucleotide identity (ANI) of 89%. Taking about 98.7% 16S identity and 95–96% ANI as the conventional species thresholds, the best conclusion is that they are
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Answer: C — different species, since high 16S identity cannot prove species identity and ANI falls short
16S rRNA is too conserved to resolve close species: identity above 98.7% is necessary but not sufficient for one species. The genome-wide ANI of 89% is well below 95–96%, so they are different species, very likely of the same genus given 99.2% 16S identity. The species threshold is not a genus threshold, so concluding separate genera overreaches; and genome measures, not 16S, decide species.Which of the following statements about molecular taxonomy are correct?
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Answer: A — The 16S rRNA gene has conserved regions that bind universal primers and variable regions that distinguish taxa; B — DNA–DNA hybridisation of 70% or more was the classic criterion for placing strains in one species
The mosaic of conserved and variable regions is what makes 16S rRNA useful, and 70% DDH was the long-standing species boundary. Very different genomes can have the same overall G+C, so equal G+C does not imply relatedness (though a large difference rules it out). An unrooted tree shows only branching relationships; direction and ancestry need a root, usually from an outgroup.