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Microbiology is the branch of biology that studies microorganisms, which are organisms too small to be seen clearly without magnification, together with viruses and other acellular infectious agents. The cellular microorganisms include the domains Bacteria and Archaea and the microscopic members of Eukaryota, such as fungi (yeasts and molds), microalgae, and protists (including protozoa). Acellular agents studied alongside them include viruses, viroids, satellites, and infectious proteins called prions.

Early microbiology focused on the causes of infectious diseases. Molecular phylogenetics, pioneered by Carl Woese through comparative sequencing of the small-subunit ribosomal RNA (16S rRNA in prokaryotes and 18S rRNA in eukaryotes), and metagenomics have since shown that pathogens make up only a small fraction of microbial diversity. Microorganisms account for most of the cells on Earth. Bacteria alone are estimated to make up roughly 15 percent of the biosphere's biomass, second only to plants. Microbes also contain most of the genetic and metabolic diversity of life, drive the global biogeochemical cycles of carbon, nitrogen, and sulfur, and shape the physiology and immune function of their multicellular hosts.

Discovery of Microorganisms, Pure Culture, and the Three-Domain System

Antonie van Leeuwenhoek first described bacteria and protists with single-lens microscopes in the 1670s. The refutation of spontaneous generation by Louis Pasteur in the 1860s, together with the work of Robert Koch on anthrax (1876) and tuberculosis (1882), established the germ theory of disease. Koch and Friedrich Loeffler formalized criteria for attributing a disease to a microbe, known as Koch's postulates (1884). The postulates have well-known limits. Some pathogens cannot be grown in pure culture, some infections are asymptomatic in healthy carriers, and many diseases involve more than one microbe.

Technical advances made pure cultures possible. They included solid media gelled with agar, the Petri dish (1887), and the Gram stain, which was devised by Hans Christian Gram in 1884. Sergei Winogradsky and Martinus Beijerinck developed enrichment culture and showed that microbes can obtain energy from inorganic compounds, which established the concept of chemolithotrophy. Ivanovsky (1892) and Beijerinck (1898) showed that a filterable agent, later identified as tobacco mosaic virus, could transmit plant disease, which began the study of viruses. In 1977, Woese and George Fox used rRNA sequences to define the Archaea as a lineage separate from the Bacteria. The three-domain system was proposed in 1990.

Ribosomal RNA Phylogenomics and the Great Plate Count Anomaly

The three-domain system places life in Bacteria, Archaea, and Eukaryota. The term prokaryote describes cells that lack a membrane-enclosed nucleus. It is a grade of organization and not a clade, because Bacteria and Archaea are distinct lineages. Many recent phylogenomic analyses support a two-domain tree in which eukaryotes arose from within the Archaea, most closely related to the Asgard archaea. The first Asgard archaeon to be grown in culture, Prometheoarchaeum syntrophicum, was described in 2020.

Prokaryotic species are commonly delineated using genome-wide average nucleotide identity (a threshold of about 95 to 96 percent) and, historically, 16S rRNA gene similarity (a threshold of about 98.65 percent). Most microbial lineages have never been cultivated, a discrepancy known as the "great plate count anomaly". Culture-independent sequencing has revealed major uncultured groups, such as the candidate phyla radiation, a large bacterial radiation of small cells with reduced genomes that is thought to depend on other organisms. Phylum names such as Bacillota (formerly Firmicutes), Bacteroidota, Actinomycetota, and Pseudomonadota (formerly Proteobacteria) have been validly published under the International Code of Nomenclature of Prokaryotes, and genome-based taxonomies such as the Genome Taxonomy Database are widely used. Viruses are classified separately by the International Committee on Taxonomy of Viruses.

Nucleoid Organization, 70S Ribosomes, and Cytoskeletal Homologs

Prokaryotes lack a nucleus. Their chromosome, usually a single circular DNA molecule, is organized in a cytoplasmic region called the nucleoid, although linear chromosomes occur in some genera such as Borrelia and Streptomyces. Their ribosomes are 70S particles composed of 30S and 50S subunits. Prokaryotes are not structureless. They have cytoskeletal homologs of eukaryotic proteins, including FtsZ (a tubulin homolog that forms the division ring) and MreB (an actin homolog that directs cell-wall synthesis in rod-shaped cells). Many contain protein-bounded organelles such as carboxysomes and lipid-bounded organelles such as magnetosomes. The physiology of a prokaryote, its classification, and its susceptibility to antimicrobial agents depend largely on the architecture of its cell envelope.

Peptidoglycan and the Gram stain

Almost all bacteria except the wall-less Mollicutes (such as Mycoplasma) have a peptidoglycan (murein) sacculus that resists internal osmotic turgor pressure. This pressure is about 3 atmospheres in Gram-negative bacteria and can reach about 20 atmospheres or more in Gram-positive bacteria. Members of the Chlamydiae have an outer membrane and little detectable peptidoglycan except at their division septum. Peptidoglycan is a mesh of glycan strands made of alternating _β_-(1→4)-linked _N_-acetylglucosamine (NAG) and _N_-acetylmuramic acid (NAM). A short stem peptide is attached to the lactoyl group of each NAM. In the pentapeptide precursor it is L-alanine, D-glutamate, a diamino acid (_meso_-diaminopimelic acid in most Gram-negative bacteria and many Bacillota rods, or L-lysine in many Gram-positive cocci), and a terminal D-alanyl-D-alanine. The D-amino acids make the wall resistant to most host peptidases.

Glycan strands are extended by transglycosylases, which include the SEDS proteins RodA and FtsW. Strands are cross-linked by penicillin-binding proteins (PBPs), which are D,D-transpeptidases. A PBP removes the terminal D-alanine from one stem peptide and joins the penultimate D-alanine to the diamino acid of a neighboring peptide, either directly (as in Escherichia coli) or through an interpeptide bridge (a pentaglycine bridge in Staphylococcus aureus). Mycobacteria also use L,D-transpeptidases to form 3→3 cross-links. Beta-lactam antibiotics (penicillins, cephalosporins, and carbapenems) mimic the acyl-D-alanyl-D-alanine substrate and acylate the active-site serine of PBPs, which irreversibly inhibits them. Glycopeptides such as vancomycin act differently, by binding the D-Ala-D-Ala terminus of the substrate itself.

The envelope architecture divides bacteria into two main classes, with some variants:

• Gram-positive bacteria (monoderms): These have one cytoplasmic phospholipid bilayer surrounded by a thick peptidoglycan layer (about 20 to 80 nm). Anionic polymers of glycerol phosphate or ribitol phosphate called teichoic acids run through the wall. Wall teichoic acids are covalently attached to NAM, and lipoteichoic acids are anchored in the cytoplasmic membrane. They bind divalent cations, regulate autolysin activity, and contribute to cell-surface charge. In the Gram stain, ethanol or acetone dehydrates the thick peptidoglycan and shrinks its pores, so that the crystal violet-iodine complex is retained and the cells look purple.
• Gram-negative bacteria (diderms): These have a cytoplasmic membrane and a bacterial outer membrane, separated by an aqueous periplasm that contains a thin peptidoglycan layer (about 2 to 7 nm). The outer membrane is asymmetric. Its inner leaflet is composed of phospholipids, and its outer leaflet is composed mostly of lipopolysaccharide (LPS), which is delivered to the surface by the Lpt protein bridge. The lipoprotein Lpp (Braun's lipoprotein) tethers the outer membrane covalently to peptidoglycan. The Mla pathway helps maintain the asymmetry of the membrane. LPS has three parts:
• Lipid A: A phosphorylated glucosamine disaccharide with several acyl chains (six in E. coli) that anchors LPS in the membrane and acts as an endotoxin. Even very low concentrations of lipid A are recognized by Toll-like receptor 4 (TLR4) and its partner MD-2 on macrophages and other cells, which triggers release of TNF-_α_, IL-1_β_, and IL-6. Overwhelming stimulation can lead to disseminated intravascular coagulation and septic shock.
• Core oligosaccharide: A branched oligosaccharide containing unusual sugars such as 3-deoxy-D-_manno_-oct-2-ulosonic acid (Kdo) and heptoses. Its phosphate groups are bridged by Mg²⁺ and Ca²⁺, which makes the outer leaflet a tight barrier to hydrophobic antibiotics and bile salts.
• O antigen: A variable polysaccharide that extends from the cell surface. It protects against the complement system and varies in structure from strain to strain, which is the basis of serotyping.

• Mycobacterial envelope: In the Mycobacteriales (including Mycobacterium, Corynebacterium, and Nocardia), peptidoglycan is covalently linked to a branched arabinogalactan polysaccharide, which is esterified to mycolic acids. In mycobacteria these long _α_-alkyl, _β_-hydroxy fatty acids have chains of about 60 to 90 carbons. They form a waxy outer "mycomembrane" that accounts for the poor Gram staining of mycobacteria, the need for hot carbol fuchsin in the acid-fast stain, and high intrinsic resistance to many hydrophilic drugs and to desiccation.

Archaeal cell envelopes

The informational machinery of Archaea resembles that of eukaryotes. Archaeal RNA polymerases are homologous to eukaryotic RNA polymerase II, transcription initiation uses a TATA-binding protein and a TFIIB homolog, and many archaea have histones. Their membranes differ completely from bacterial and eukaryotic membranes, a difference called the "lipid divide". Archaeal membrane lipids are built from branched isoprenoid chains joined by chemically stable ether bonds to _sn_-glycerol-1-phosphate. Bacterial and eukaryotic lipids use unbranched fatty acids joined by ester bonds to the enantiomer _sn_-glycerol-3-phosphate. Many archaea, including methanogens and halophiles, make bilayers of 20-carbon diether lipids (archaeol). Many thermophiles and acidophiles, such as Sulfolobus, also have membrane-spanning 40-carbon tetraether lipids (caldarchaeol and related glycerol dialkyl glycerol tetraethers), often with cyclopentane rings. These form a covalent monolayer that has low proton permeability and tolerates high temperature and strongly acidic conditions. Hyperthermophilic archaea grow above 80 °C, and the highest growth temperature recorded is about 122 °C, for Methanopyrus kandleri strain 116 at high pressure. Some acidophiles such as Picrophilus grow near pH 0.

Archaea lack bacterial peptidoglycan, so they are insensitive to _β_-lactams. Most archaea are coated with an S-layer, a paracrystalline array of glycoprotein subunits. Methanogens of the orders Methanobacteriales and Methanopyrales have pseudopeptidoglycan (pseudomurein), a polymer of alternating NAG and _N_-acetyltalosaminuronic acid linked by _β_-(1→3) bonds with L-amino acid peptide cross-links. It is insensitive to lysozyme. Archaeal cell division uses FtsZ in the Euryarchaeota, whereas in many Crenarchaeota it uses a system related to the eukaryotic ESCRT-III machinery (Cdv proteins).

Endospores

Some Bacillota, including Bacillus and Clostridium, form endospores when nutrients are depleted. The spore has a dehydrated core with a high content of calcium dipicolinate, small acid-soluble proteins that bind and protect the DNA, and a thick cortex and coat. It tolerates heat, desiccation, radiation, and many disinfectants, and can remain viable for years. When nutrients return, specific germinant receptors trigger germination (see the discussion of Clostridioides difficile below).

Growth kinetics

Most bacteria reproduce by binary fission. The population in balanced growth in a batch culture increases exponentially. If N₀ is the initial cell number and t_d is the doubling time, then:

Here μ is the specific growth rate. A batch culture passes through a lag phase, an exponential phase, a stationary phase (when a nutrient is depleted or waste products accumulate), and a death phase. Escherichia coli can double every 20 minutes or so in rich medium at 37 °C. This is shorter than the time needed to replicate its chromosome (about 40 minutes) plus the time from termination to division (about 20 minutes), because cells initiate new rounds of replication before earlier rounds finish (multifork replication).

At a limiting nutrient concentration S, growth rate is often described by the Monod equation:

where μ_max is the maximum specific growth rate and K_s is the concentration of substrate at which μ is half of μ_max. In a chemostat, fresh medium enters at a flow rate F into a vessel of volume V, and the dilution rate is D = _F_/_V_. At steady state μ = D, so the residual substrate concentration is:

The culture is washed out if D exceeds the maximum growth rate sustainable at the inflowing substrate concentration.

Metabolic diversity

Microorganisms are classified by their sources of energy and carbon:

Electrons from the oxidation of the energy source can be passed to O₂ in aerobic respiration, to other terminal acceptors such as nitrate, sulfate, Fe(III), or CO₂ in anaerobic respiration, or to an organic compound derived from the substrate in fermentation. Microbes carry out several processes with no direct counterpart in animals and plants, including biological nitrogen fixation by nitrogenase (an oxygen-sensitive enzyme that consumes at least 16 ATP per N₂, protected in some cyanobacteria by heterocysts and in legume nodules by leghemoglobin), nitrification, denitrification, anammox (anaerobic oxidation of ammonium with nitrite, carried out by Planctomycetota), and methanogenesis.

Environmental limits

Microorganisms are classified by growth temperature as psychrophiles (optimum below about 15 °C), mesophiles (about 20 to 45 °C), thermophiles (about 45 to 80 °C), and hyperthermophiles (above 80 °C). They also differ in tolerance of pH (acidophiles and alkaliphiles), salinity (halophiles), pressure (piezophiles), and radiation. Deinococcus radiodurans tolerates ionizing radiation doses that are lethal to most organisms, a tolerance attributed to efficient DNA repair and protection of proteins from oxidative damage. Collectively these organisms are called extremophiles.

Fluid mechanics at low Reynolds number

The physical environment of a swimming microorganism is described by the Reynolds number (Re), the ratio of inertial to viscous forces:

where ρ is the fluid density (about 10³ kg/m³ for water), v is the swimming speed (about 20 to 50 μm/s), L is the cell size (about 1 to 2 μm), and η is the dynamic viscosity (about 10⁻³ Pa·s). For a swimming bacterium Re is roughly 10⁻⁵ to 10⁻⁴. In this regime of Stokes flow, inertia is negligible. For a sphere of radius a the drag force is F = 6π_η a v_ (Stokes' law), and a bacterium that stops propelling itself coasts for less than a microsecond, over a distance of the order of a tenth of an ångström. Edward Mills Purcell showed that under these conditions any motion that is the same forward and in reverse produces no net displacement, a result known as the scallop theorem. Microbes therefore swim with non-reciprocal motions, most commonly by rotating a helical filament like a corkscrew.

The bacterial flagellar motor

The bacterial flagellum is a rotary nanomachine of several megadaltons that spans the cell envelope. It is built from about 40 to 50 gene products expressed in a regulated hierarchy. It has three parts:

1. Basal body: The rotor and its bearings. The cytoplasmic C-ring (FliG, FliM, and FliN) is the switch complex. The MS-ring (FliF) sits in the inner membrane. In Gram-negative bacteria, the P-ring and L-ring act as bushings in the peptidoglycan and outer membrane around a central drive rod.
2. Hook: A flexible coupling (FlgE) that acts as a universal joint between the motor and the filament.
3. Filament: A hollow helical tube, usually several to 15 μm long and about 20 nm wide, made of about 20,000 subunits of flagellin (FliC). Flagellin is exported through the central channel (about 2 nm wide) by a type III secretion system at the base of the flagellum and assembles beneath a FliD cap at the distal tip.

The motors of the bacterial flagellum and the archaellum are fundamentally different from the eukaryotic flagellum, which is moved by dynein ATPases sliding the microtubules of a 9+2 axoneme. The archaeal archaellum is related to type IV pili and is driven by ATP hydrolysis (the ATPase FlaI), whereas the bacterial flagellar motor is driven by an ion gradient across the cytoplasmic membrane.

In Escherichia coli and Salmonella enterica, the energy source is the proton-motive force (Δp), which is the sum of an electrical and a chemical term:

where Δψ = ψ_in − ψ_out, _Δ_pH = pH_in − pH_out, R is the gas constant, T is the absolute temperature, and F is the Faraday constant. At 30 °C, 2.303 _RT_/_F_ is about 60 mV per pH unit, and Δp is typically about −140 to −200 mV. The motor has up to about 11 stator units, each a MotA₅MotB₂ complex anchored to the peptidoglycan by MotB. Protons flow through channels in the stator, and the protonation and deprotonation of a conserved aspartate (Asp32 of MotB in E. coli) drives rotation of the MotA ring around the MotB dimer, which applies torque to FliG of the rotor. The motor uses about 1,200 protons per revolution and rotates at about 100 to 300 Hz in E. coli.

In marine Vibrio species such as Vibrio alginolyticus and Vibrio cholerae, the polar flagellum is driven by a sodium-motive force through PomA₅PomB₂ stators. These motors can reach frequencies of about 1,700 Hz (about 100,000 revolutions per minute) and propel cells at over 100 μm/s.

Other forms of motility

Bacteria move by other mechanisms as well. Type IV pili extend, attach to a surface, and retract to pull cells in "twitching" motility. In Myxococcus xanthus, gliding motility depends on focal-adhesion-like complexes and a pili-based system. In spirochetes, flagella lie within the periplasm and rotate to flex the whole cell. Magnetotactic bacteria align with magnetic field lines using chains of magnetosomes.

Chemotaxis

A bacterium is too small to compare concentrations reliably across its own length, so it measures a gradient by comparing concentrations over time. It moves by a biased random walk of "runs" and "tumbles", controlled by a two-component signaling pathway:

• Run: When flagella rotate counterclockwise (viewed from outside the cell), the left-handed filaments of a peritrichous cell form a coordinated bundle and the cell swims in a straight line for about a second.
• Tumble: When one or more motors switch to clockwise rotation, their filaments undergo a polymorphic transformation to a different helical form and leave the bundle. The cell then tumbles for about 0.1 seconds and reorients randomly.

Transmembrane methyl-accepting chemotaxis proteins (MCPs such as Tar and Tsr), which form arrays at the cell poles with the histidine kinase CheA and the adaptor CheW, detect attractants and repellents. Binding of an attractant inhibits CheA, and a decrease in attractant occupancy increases CheA activity. CheA autophosphorylates on a histidine and passes the phosphoryl group to an aspartate of the response regulator CheY. Phosphorylated CheY (CheY-P) diffuses to the switch complex, binds FliM, and promotes clockwise rotation (a tumble). When a cell swims up a gradient, receptor occupancy rises, CheA activity falls, and CheZ rapidly dephosphorylates CheY-P, so runs become longer. When a cell swims down a gradient, CheA activity rises and tumbles become more frequent.

Adaptation restores sensitivity over a wide range of background concentrations. The methyltransferase CheR continuously methylates glutamate residues of the MCPs (using _S_-adenosylmethionine), which raises CheA activity, and the methylesterase CheB, which is activated by phosphorylation by CheA, removes the methyl groups. This forms an integral feedback loop that gives the cell a memory of the past few seconds.

Mutation and gene regulation

The fluctuation test of Luria and Delbrück (1943) showed that mutations conferring phage resistance arise at random before selection and are not induced by it. Replication of the E. coli chromosome begins when DnaA unwinds oriC. Chromosome replication fork progression is about 1,000 nucleotides per second, and division requires the tubulin homolog FtsZ. Bacterial genes are often organized in operons, as first shown for the lac operon by François Jacob and Jacques Monod. Promoter recognition depends on sigma factors that associate with RNA polymerase, and gene expression is further controlled by riboswitches, small regulatory RNAs (often with the chaperone Hfq), and two-component systems made of a sensor histidine kinase and a response regulator.

Because prokaryotes reproduce asexually, clonal populations do not undergo meiotic recombination. Horizontal gene transfer (HGT) drives genome evolution, metabolic innovation, and the spread of antibiotic resistance. A bacterial species' genome can be divided into a core genome shared by all strains and an accessory genome carried on plasmids, transposons, integrons, and chromosomal genomic islands (including pathogenicity islands). Together they make up the pan-genome.

Mechanisms of horizontal gene transfer

1. Transformation: Uptake of free DNA from the environment. Naturally competent species include Streptococcus pneumoniae, Bacillus subtilis, Vibrio cholerae, and Neisseria gonorrhoeae. A type IV pilus or pilus-like structure binds double-stranded DNA, and a nuclease degrades one strand (EndA in S. pneumoniae) while the other strand is imported through the ComEC channel. DprA then loads the recombinase RecA, which promotes homologous recombination into the chromosome.
2. Transduction: Bacteriophage-mediated transfer of host DNA. In generalized transduction, phages that package DNA by a "headful" mechanism, such as P1 and P22, occasionally package fragments of host DNA, and these can recombine into a recipient. In specialized transduction, imprecise excision of a prophage such as phage λ carries adjacent host genes (such as gal or bio) into the progeny virions. Gene transfer agents, which are phage-like particles that package random host DNA, are an additional route in some bacteria and archaea.
3. Conjugation: Contact-dependent transfer of DNA from a donor to a recipient, mediated by conjugative plasmids (such as the F plasmid) or integrative and conjugative elements. A type IV secretion system forms a pilus that attaches to the recipient and retracts. A relaxosome assembles at the origin of transfer (oriT), and the relaxase (TraI in F) cuts one strand at nic and remains covalently attached to its 5′ end through a tyrosine. The relaxase and the strand are pumped through the T4SS into the recipient, where the relaxase re-circularizes the strand and host DNA polymerase III synthesizes the complement. The donor replicates its remaining strand by rolling circle replication.

Prokaryotic defense systems and CRISPR-Cas

Bacteria and archaea are constantly attacked by phages and other mobile elements. Genes for defense systems are often clustered in "defense islands", and hundreds of distinct systems have been described. Examples include:

• Restriction-modification systems: A methyltransferase modifies host DNA at a recognition sequence, and a cognate restriction enzyme cuts unmethylated foreign DNA with the same sequence.
• Abortive infection and signaling systems: In these systems, infection triggers suicide of the infected cell, which prevents the phage from spreading to kin. Examples include CBASS (cyclic-oligonucleotide-based antiphage signaling systems), which use cGAS-like nucleotidyltransferases related to the mammalian cGAS-STING pathway, and the Thoeris system, in which TIR-domain proteins deplete NAD⁺. Others include retrons, Gabija, and toxin-antitoxin modules.
• CRISPR-Cas adaptive immunity: About 40 percent of sequenced bacterial genomes and a majority of archaeal genomes encode a CRISPR-Cas system. A CRISPR array of repeats separated by short spacers derived from past invaders provides a heritable record of infections. Immunity has three stages:
• Adaptation: The Cas1-Cas2 complex takes a short fragment (a protospacer, often next to a short protospacer adjacent motif, or PAM) from foreign DNA and integrates it as a new spacer at the leader end of the array.
• Expression and processing: The array is transcribed into a precursor CRISPR RNA (pre-crRNA), which is cleaved into short crRNAs. In type II systems the cleavage depends on a trans-activating tracrRNA and host RNase III, and in other types it depends on Cas6 or related enzymes.
• Interference: Class 1 systems (types I, III, and IV) use multi-subunit effector complexes, such as Cascade with the Cas3 nuclease-helicase in type I. Class 2 systems (types II, V, and VI) use a single large effector protein such as Cas9, Cas12a, or the RNA-targeting Cas13. The crRNA guides the effector to a complementary target, which for DNA targets in many systems also requires a PAM, and the invader is cleaved or silenced.

Antibacterial Targets and Biochemical Mechanisms of Resistance

Antibiotics act on bacterial processes that differ from those of their hosts. The main targets are:

• Cell wall synthesis: beta-lactams (inhibit PBPs) and glycopeptides such as vancomycin (bind D-Ala-D-Ala).
• Protein synthesis: aminoglycosides, tetracyclines (30S subunit); macrolides, chloramphenicol, and linezolid (50S subunit).
• Nucleic acid synthesis: fluoroquinolones (DNA gyrase and topoisomerase IV) and rifampicin (RNA polymerase).
• Folate metabolism: sulfonamides and trimethoprim.
• Membranes: polymyxins and daptomycin.

Bacteria resist antibiotics by several mechanisms. Resistance genes are often carried on plasmids or other mobile elements, so they spread by HGT.

• Enzymatic inactivation of the drug, for example by beta-lactamases (including extended-spectrum enzymes and carbapenemases such as KPC and NDM) and aminoglycoside-modifying enzymes.
• Target alteration or replacement. Staphylococcus aureus acquires mecA, which encodes a PBP (PBP2a) with low affinity for most beta-lactams (methicillin-resistant S. aureus, or MRSA). The vanA operon changes the peptidoglycan precursor terminus to D-alanyl-D-lactate, which reduces vancomycin binding. Mutations in gyrA reduce fluoroquinolone binding, and Erm methyltransferases methylate 23S rRNA to block macrolides.
• Efflux by pumps such as the AcrAB-TolC system of E. coli, and reduced permeability through loss or alteration of porins.
• Target protection by proteins that dislodge a drug from the ribosome, such as Tet(M).

Resistance (an increased minimum inhibitory concentration) should be distinguished from tolerance (survival of a whole population for longer during killing) and persistence (survival of a small subpopulation, discussed below). Antimicrobial resistance is a major health problem. Bacterial infections caused by resistant organisms were estimated to account directly for about 1.1 million deaths worldwide in 2021, with substantially more deaths associated with resistance.

Microorganisms in nature live in interdependent communities. In syntrophy, one organism consumes the waste products of another, as in the interspecies transfer of H₂ from fermenting bacteria to methanogens, which keeps H₂ concentrations low enough for fermentation to remain thermodynamically favorable. Microbes also drive the cycles of nitrogen (fixation, nitrification, denitrification, anammox), sulfur, and carbon. In the oceans, viruses (mostly bacteriophages) are estimated to lyse a substantial fraction of bacterial cells each day (often estimated at 20 to 40 percent of production). The lysis releases organic matter that is recycled by other microbes, a process called the viral shunt.

The human body is a holobiont that hosts about 4 × 10¹³ microbial cells (a recent estimate is 3.8 × 10¹³), which is about the same as the number of human cells (a ratio of roughly 1.3 to 1). The collective genes of this microbiome outnumber the roughly 20,000 protein-coding genes of the human genome by about two orders of magnitude.

Anatomical niches

Body sites differ in pH, oxygen, moisture, and antimicrobial secretions, and each selects its own community.

• Skin: The skin surface is dry, salty, and mildly acidic (pH about 4.5 to 5.5), owing to sweat, free fatty acids produced from sebum by lipophilic microbes such as Cutibacterium acnes within hair follicles, and the breakdown products of epidermal proteins. Exposed skin is dominated by salt-tolerant Gram-positive cocci, including Staphylococcus epidermidis. Commensal strains of S. epidermidis reinforce the skin barrier. A sphingomyelinase of S. epidermidis produces ceramides, phenol-soluble modulins act together with host peptides such as LL-37 to kill S. aureus and Streptococcus pyogenes, and TLR2 signaling induces keratinocytes to produce antimicrobial peptides. Other commensals produce bacteriocins and related inhibitors, such as lugdunin, a cyclic nonribosomal peptide made by Staphylococcus lugdunensis that inhibits S. aureus. Through such mechanisms, commensals contribute to competitive exclusion of pathogens.
• Gastrointestinal tract: The stomach (pH about 1.5 to 3) and the bile-rich duodenum have sparse communities (about 10¹ to 10⁴ bacteria per mL). Bacterial density rises along the small intestine and reaches 10¹¹ to 10¹² cells per gram of content in the anoxic colon. Colonic communities are dominated by obligate anaerobes of the phyla Bacteroidota and Bacillota, with substantial contributions from Actinomycetota (for example Bifidobacterium), Verrucomicrobiota (Akkermansia muciniphila), and methanogenic archaea (Methanobrevibacter smithii).

Fermentation of fiber and short-chain fatty acids

Humans encode only a small number of digestive glycoside hydrolases, which cleave starch, sucrose, and lactose. Complex plant polysaccharides such as cellulose, hemicellulose, pectin, inulin, and resistant starches therefore reach the colon undigested. The colonic microbiome encodes thousands of carbohydrate-active enzymes. In Bacteroides, these are organized in polysaccharide utilization loci, such as the Sus system for starch, in which surface proteins bind glycans, a surface endoglycanase cleaves them, and TonB-dependent transporters (SusC) import the oligosaccharides into the periplasm.

Fermentation of fiber yields mainly the short-chain fatty acids acetate, propionate, and butyrate, at molar ratios of roughly 60:20:20, and total concentrations of about 50 to 150 mM in the colon.

• Metabolism: Acetate and propionate pass into the portal blood. Propionate is a substrate for hepatic gluconeogenesis, and acetate is a substrate for lipogenesis. Butyrate is the main energy source of colonocytes and supplies a large share of their ATP by beta oxidation. The resulting high oxygen consumption keeps the epithelial surface hypoxic, which limits the oxygen and nitrate available to facultative anaerobic Enterobacteriaceae (such as pathogenic Escherichia and Salmonella).
• Immune effects: Short-chain fatty acids signal through G protein-coupled receptors (GPR43/FFAR2, GPR41/FFAR3, and GPR109A/HCAR2) on epithelial and immune cells. Butyrate also inhibits histone deacetylases. In naive CD4⁺ T cells this increases acetylation of histone H3 at the FOXP3 locus and promotes differentiation of peripheral regulatory T cells, which secrete interleukin 10 and TGF-_β_ and help maintain tolerance to the microbiota.

Bile acids, dysbiosis, and colonization resistance

A healthy microbiome resists colonization by pathogens by consuming limiting nutrients (monosaccharides, sialic acid, iron), by stimulating host antimicrobial responses (RegIII_γ_ and secretory IgA), and by modifying bile acids. The liver converts cholesterol into the primary bile acids cholic acid and chenodeoxycholic acid, which are conjugated to taurine or glycine. About 95 percent is reabsorbed in the ileum. The remaining 5 percent reaches the colon, where bile salt hydrolases deconjugate it. A limited group of anaerobes, such as Clostridium scindens, carry the bai operon and perform 7_α_-dehydroxylation to form the secondary bile acids deoxycholic acid and lithocholic acid.

Broad-spectrum antibiotics such as clindamycin, fluoroquinolones, and third-generation cephalosporins can eliminate these anaerobes and cause dysbiosis. Taurocholate then accumulates, and secondary bile acids that inhibit vegetative growth disappear. Taurocholate is a germinant of Clostridioides difficile spores, which sense it through a Csp protease-like receptor system. Vegetative cells secrete the large glucosylating toxins TcdA and TcdB, which transfer glucose to a threonine of Rho, Rac, and Cdc42 GTPases and cause collapse of the actin cytoskeleton, loss of tight junctions, and pseudomembranous colitis. Recurrent infection can be treated by restoring the microbiota, for example by fecal microbiota transplantation or by approved microbiome-based products.

A pathogen's ability to evade immunity, replicate in host tissue, and transmit depends on virulence factors. Many of these are encoded on mobile elements, so virulence can spread by HGT.

Secretion systems

Bacteria export proteins with nine classes of secretion systems (types I to IX). Important examples include:

• Type III secretion system (T3SS, injectisome): This syringe-like apparatus is evolutionarily related to the flagellar basal body. It is found in Gram-negative pathogens such as Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa, and enteropathogenic E. coli. After contact with a host cell, a translocon pore is inserted into the host membrane, and the system, powered by an ATPase and the proton-motive force, injects effector proteins into the host cytoplasm. Salmonella effectors SopE and SopE2 are guanine nucleotide exchange factors that activate Rac1 and Cdc42 and cause actin-driven membrane ruffling and bacterial uptake, and SipA binds actin. Yersinia effectors disable host defenses. YopH is a tyrosine phosphatase, YopE is a GTPase-activating protein, and YopJ is an acetyltransferase that blocks NF-_κ_B and MAPK signaling.
• Type IV secretion system (T4SS): The T4SS is evolutionarily related to conjugation machinery. Helicobacter pylori uses it to inject the effector CagA into gastric epithelial cells, Legionella pneumophila uses the Dot/Icm system to inject more than 300 effectors that convert its phagosome into a replicative vacuole, and Agrobacterium tumefaciens uses it to transfer T-DNA into plant cells.
• Type VI secretion system (T6SS): The T6SS resembles an inverted contractile phage tail. Contraction of an outer sheath (VipA and VipB, also called TssB and TssC) drives an inner tube of Hcp, tipped with a VgrG and PAAR spike, out of the cell. It delivers toxins into neighboring bacteria or into eukaryotic cells, and it is often used in interbacterial competition.

Bacterial exotoxins

Exotoxins are proteins secreted by bacteria that act at very low concentrations. They are grouped as follows:

1. Type I (cell-surface acting), including superantigens: Staphylococcal toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxins bind outside the peptide-binding groove of MHC class II molecules and to the variable beta domain of the T-cell receptor. This cross-links many T cells regardless of antigen specificity and activates a far larger fraction (up to roughly 5 to 20 percent) than normal antigen-specific responses, which causes a cytokine storm (IFN-_γ_, TNF-_α_, IL-2).
2. Type II (membrane damaging): These include cholesterol-dependent cytolysins such as pneumolysin of S. pneumoniae and listeriolysin O of Listeria monocytogenes. They assemble into large ring-shaped prepores of dozens of subunits that insert as _β_-barrel pores, and listeriolysin O also allows escape from the phagosome. Phospholipases such as Clostridium perfringens _α_-toxin hydrolyze membrane phosphatidylcholine and sphingomyelin.
3. Type III (A-B toxins): A binding (B) domain attaches to a host receptor and mediates uptake, and a catalytic (A) subunit enters the cytosol.

• Inhibition of protein synthesis: Diphtheria toxin (encoded by a prophage in Corynebacterium diphtheriae) and Pseudomonas exotoxin A transfer ADP-ribose from NAD⁺ to diphthamide, a modified histidine of eukaryotic elongation factor 2, which halts translation. Shiga toxin (from Shigella dysenteriae and Shiga toxin-producing E. coli) is an _N_-glycosidase that removes an adenine from the sarcin-ricin loop of 28S rRNA.
• Dysregulation of second messengers: Cholera toxin (and the related heat-labile toxin of enterotoxigenic E. coli) has an AB₅ structure. The B pentamer binds GM1 ganglioside, and the A₁ subunit ADP-ribosylates the stimulatory G protein G_α__s, which inhibits its GTPase activity. Adenylate cyclase remains active, cAMP rises, and protein kinase A activates the CFTR chloride channel and inhibits Na⁺/H⁺ exchange. The result is a secretory diarrhea with fluid loss that can reach 15 to 20 liters per day.
• Blockade of neurotransmitter release: Botulinum toxin (Clostridium botulinum) and tetanus toxin (Clostridium tetani) are zinc metalloproteases that cleave SNARE proteins. Botulinum toxin serotypes cleave SNAP-25, synaptobrevin, or syntaxin and block release of acetylcholine at neuromuscular junctions, which causes flaccid paralysis. Tetanus toxin travels in retrograde fashion to the central nervous system, where it cleaves synaptobrevin in inhibitory interneurons. This blocks release of glycine and GABA and causes spastic paralysis.

Quorum sensing, cyclic di-GMP, and biofilms

In quorum sensing, bacteria secrete diffusible signals called autoinducers and respond when the concentration reaches a threshold, which usually reflects high cell density and restricted diffusion. Main systems include:

• LuxI/LuxR systems of Gram-negative bacteria (for example Vibrio fischeri and Pseudomonas aeruginosa): LuxI-type synthases make _N_-acyl homoserine lactones, which bind cytoplasmic LuxR-type transcription factors.
• Peptide systems of Gram-positive bacteria: In Staphylococcus aureus the agr locus encodes a processed thiolactone autoinducing peptide, exported by the membrane protein AgrB, which is sensed by the histidine kinase AgrC and the response regulator AgrA.
• AI-2: The enzyme LuxS produces 4,5-dihydroxy-2,3-pentanedione (DPD), which converts to autoinducer-2 in several forms (one is a borate complex in Vibrio harveyi). AI-2 is made by many species and has been proposed as an interspecies signal, but its role in many bacteria is debated.

The second messenger cyclic di-GMP controls the switch between motile and sessile lifestyles. It is made from two GTP molecules by diguanylate cyclases (GGDEF domains) and degraded by phosphodiesterases (EAL or HD-GYP domains). Elevated c-di-GMP binds PilZ-domain proteins and riboswitches, inhibits motility, and promotes production of the matrix.

A biofilm is a community of cells embedded in a self-produced matrix of extracellular polymeric substances. The matrix contains exopolysaccharides (Pel, Psl, and alginate in P. aeruginosa; poly-_β_-1,6-_N_-acetylglucosamine in S. aureus and S. epidermidis), functional amyloids (curli in E. coli, TasA in B. subtilis), and extracellular DNA, and it is interlaced with water channels. Biofilms cause chronic infections such as P. aeruginosa in the airways in cystic fibrosis and S. aureus on prosthetic devices. Cells in biofilms tolerate antibiotics at 10 to 1,000 times the concentrations that kill planktonic cells. The matrix can retard diffusion of some drugs (as alginate does for aminoglycosides), gradients of oxygen and nutrients produce slow-growing cells, and a subpopulation of persister cells enters a transient, drug-tolerant state. Many antibiotics depend on active growth or metabolism (cell-wall synthesis for _β_-lactams, a proton-motive force for aminoglycoside uptake, and DNA replication for fluoroquinolones), so persisters survive and can regrow after treatment stops.

Viruses are acellular obligate intracellular parasites. Virions lack ribosomes and the capacity to make ATP, and outside a host they are inert particles that protect and deliver a nucleic acid genome. Some giant viruses encode components of the translation machinery but no complete ribosome.

Virion structure and capsid geometry

A virion consists of a DNA or RNA genome inside a protein shell, the capsid, built from repeated subunits. Genome sizes range from about 1.7 kb in circoviruses to about 2.5 Mb in Pandoravirus giant viruses. Because the genome has limited coding capacity, Crick and Watson proposed in 1956 that a capsid is built from many copies of one or a few small proteins arranged symmetrically.

• Helical capsids form when protein subunits bind along the nucleic acid, as in tobacco mosaic virus. The nucleocapsids of influenza A virus, rabies virus, and filoviruses are also helical.
• Icosahedral capsids are built on an icosahedron, which has 20 triangular faces, 12 five-fold vertices, 30 two-fold edges, and 20 three-fold axes. A simple icosahedron holds 60 identical subunits. Larger capsids follow the Caspar-Klug theory of quasi-equivalence, in which the subunits occupy slightly different environments, and the number of subunits is 60_T_, where the triangulation number T is:

Here h and k are non-negative integers that define the spacing of the five-fold vertices on the hexagonal lattice. The permitted values of T are 1, 3, 4, 7, 9, 12, 13, 16, 19, 21, 25, and so on. Such a capsid always has 12 pentamers at the vertices and 10(T − 1) hexamers. Parvoviruses have T = 1, the bacteriophage MS2 has T = 3, papillomaviruses and polyomaviruses have T = 7, and herpesvirus capsids have T = 16.

Non-enveloped viruses, such as picornaviruses, adenoviruses, and noroviruses, depend on the capsid to survive desiccation and the digestive tract. Enveloped viruses, such as coronaviruses, orthomyxoviruses, paramyxoviruses, flaviviruses, herpesviruses, and retroviruses, are surrounded by a lipid bilayer derived from a host membrane (the plasma membrane, endoplasmic reticulum, Golgi apparatus, or nuclear membrane) that is studded with viral glycoproteins used for attachment and membrane fusion.

The Baltimore classification

Cellular organisms store genetic information in double-stranded DNA, whereas viral genomes can be DNA or RNA, single-stranded or double-stranded, linear, circular, or segmented. David Baltimore proposed in 1971 a classification based on how a virus makes messenger RNA (mRNA) that host ribosomes can read. His scheme originally had six classes, and a seventh, for the DNA reverse-transcribing viruses, was added later.

Replication cycle and innate sensing

The productive cycle of a virus has six stages:

1. Attachment: Viral proteins bind specific host receptors and co-receptors, which determines the tropism of the virus. Examples are SARS-CoV-2 spike binding to ACE2, HIV gp120 binding to CD4 and CCR5 or CXCR4, and influenza hemagglutinin binding to sialic acids (_α_-2,6-linked in the human upper airway and _α_-2,3-linked in avian hosts).
2. Entry: Enveloped viruses fuse their envelope with the plasma membrane at neutral pH (as in HIV and paramyxoviruses) or are taken up by endocytosis and fuse after the endosome acidifies (as in influenza and flaviviruses), when a low-pH-triggered conformational change in a fusion protein drives membrane fusion. Non-enveloped viruses usually use amphipathic peptides to disrupt or cross the endosomal membrane.
3. Uncoating: The capsid releases the genome in the cytosol or at the nuclear pore.
4. Genome replication and gene expression: Viruses typically express early genes (regulators and polymerases) before late genes (structural proteins). Most RNA virus polymerases lack proofreading, so RNA viruses mutate at roughly 10⁻⁶ to 10⁻⁴ substitutions per nucleotide per replication and exist as quasispecies. The exceptions are nidoviruses such as coronaviruses, which encode a 3′-to-5′ exoribonuclease (nsp14, ExoN) that provides proofreading.
5. Assembly: Genomes are packaged into procapsids or capsids.
6. Egress: Many non-enveloped viruses are released by lysis of the cell, although non-lytic exit also occurs. Enveloped viruses bud from membranes, often using the host ESCRT machinery.

Cells detect viruses through pattern recognition receptors. Endosomal Toll-like receptors detect double-stranded RNA (TLR3), single-stranded RNA (TLR7 and TLR8), and unmethylated CpG DNA (TLR9). The cytosolic helicases RIG-I (which senses RNA with a 5′ triphosphate and short double-stranded RNA) and MDA5 (which senses long double-stranded RNA) signal through MAVS. The DNA sensor cGAS makes 2′3′-cGAMP, which activates STING. These pathways converge on the kinase TBK1 and the transcription factors IRF3 and IRF7, which induce type I interferons (IFN-_α_ and IFN-_β_). Interferons signal through the JAK-STAT pathway to induce hundreds of interferon-stimulated genes. Examples include protein kinase R (PKR, which phosphorylates eIF2_α_ and blocks translation), 2′-5′-oligoadenylate synthetase (OAS, which activates RNase L to degrade RNA), and tetherin, which retains budding enveloped virions on the cell surface.

Bacteriophages

Bacteriophages infect bacteria, and they are the most abundant biological entities, with an estimated 10³¹ particles worldwide. A virulent phage follows a lytic cycle that ends in lysis of the host. A temperate phage such as λ can instead integrate as a prophage and replicate with the host chromosome (lysogeny), and it enters the lytic cycle when the host is stressed, for example by DNA damage. Prophages often carry genes that benefit the host, including the toxin genes of diphtheria and cholera and of Shiga toxin-producing E. coli. Phage therapy, the use of phages to treat bacterial infections, is being revisited because of antibiotic resistance.

Viroids, satellites, and prions

Viroids are small (about 250 to 400 nucleotides), circular, non-coding single-stranded RNAs that infect plants. In the family Pospiviroidae, replication takes place in the nucleus and uses host RNA polymerase II. In the family Avsunviroidae it takes place in chloroplasts and uses ribozyme self-cleavage. Satellites depend on a helper virus. The hepatitis D virus needs the envelope proteins of hepatitis B virus for assembly. Prions are infectious proteins that contain no nucleic acid. A misfolded, aggregation-prone form (PrP^Sc) of the host protein PrP^C converts normal PrP^C into the misfolded form. Prions cause transmissible spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy, kuru, and Creutzfeldt-Jakob disease.

What is the biochemical basis of the "lipid divide" between Bacteria and Archaea?

The lipid divide refers to the fundamental stereochemical and chemical divergence between bacterial/eukaryotic and archaeal plasma membranes. Bacteria and eukaryotes synthesize ester-linked unbranched fatty acyl chains attached to an _sn_-glycerol-3-phosphate (G3P) backbone. Conversely, Archaea utilize the opposite chiral enantiomer, _sn_-glycerol-1-phosphate (G1P), linked via chemically stable ether bonds to methyl-branched isoprenoid chains (20-carbon archaeol diethers or 40-carbon membrane-spanning caldarchaeol tetraether monolayers), conferring exceptional resistance to extreme heat and acidity.

How do colonic short-chain fatty acids (SCFAs) regulate host metabolism and regulatory T cells?

Short-chain fatty acids—primarily acetate, propionate, and butyrate at an approximate 60:20:20 molar ratio—are produced when anaerobic colonic bacteria (Bacteroidota and Bacillota) ferment indigestible dietary plant polysaccharides. Butyrate fuels colonocyte mitochondrial β-oxidation, consuming local O₂ to maintain luminal hypoxia that restricts facultative pathogens. Simultaneously, butyrate signals via G protein-coupled receptors (GPR43, GPR109A) and inhibits histone deacetylases (HDACs) at the FOXP3 locus, driving anti-inflammatory peripheral regulatory T-cell (Treg) differentiation.

Why are bacterial biofilms up to 1,000-fold more tolerant to antibiotics than planktonic cells?

Biofilm antibiotic tolerance arises from three synergistic biophysical and physiological mechanisms rather than new genetic mutations alone. First, the self-secreted extracellular polymeric substance (EPS) matrix of exopolysaccharides (such as anionic alginate) and eDNA electrostatically retards the diffusion of positively charged drugs like aminoglycosides. Second, steep inward oxygen and nutrient gradients force interior cells into stationary phase. Third, a small subpopulation differentiates into metabolically dormant persister cells whose inactive targets (PBPs, ribosomes, DNA gyrase) cannot be corrupted by bactericidal antibiotics.