Pathogens & antibiotics

The lac operon of E. coli lets the cell use lactose only when it is present and glucose is scarce. It has two levels of control: a negative one (the repressor) and a positive one (CAP-cAMP). Convention used here: gene names are written plainly in lower case (lacZ, lacY, lacA, lacI) and protein names with a capital letter (LacI, TrpR, AraC).

Negative control. With no lactose, the repressor sits on the operator and the operon is off (a very low basal level remains). When lactose enters, the few β-galactosidase molecules made at the basal level convert some of it to allolactose, the natural inducer, which binds the repressor, changes its shape and releases it from the operator. IPTG is a gratuitous inducer: it induces but is not metabolised.

Positive control and catabolite repression. Low glucose raises cAMP. The CAP (also called CRP) protein binds cAMP, and the CAP-cAMP complex binds near the promoter and helps RNA polymerase start. High glucose lowers cAMP, so CAP is inactive and the operon stays at a low level even if lactose is present. Glucose also acts by inducer exclusion: while glucose is taken up by the phosphotransferase system, the unphosphorylated EIIA(Glc) protein binds LacY and blocks lactose entry. Maximal expression therefore needs both lactose present (repressor off) and glucose absent (CAP-cAMP on).

The trp operon encodes five polypeptides (trpE, trpD, trpC, trpB, trpA) that form the enzymes building tryptophan from chorismate, among them anthranilate synthase (TrpE with TrpD) and tryptophan synthase (TrpA with TrpB). It is a repressible operon: the end product switches it off. Two layers of control act: repression and attenuation.

Repression. TrpR is made as an inactive aporepressor. When tryptophan is abundant it acts as a corepressor: it binds TrpR, which then binds the operator and blocks transcription. When tryptophan is scarce, it dissociates from TrpR, the repressor leaves the operator and transcription starts.

Attenuation. A leader region (trpL) lies between the operator and trpE. It has four stretches (1, 2, 3, 4), and region 1 encodes a short leader peptide with two adjacent tryptophan codons. The RNA can pair 1-2, 2-3 or 3-4. The 3-4 hairpin followed by a run of U's is a rho-independent terminator; the 2-3 hairpin is an anti-terminator, and it prevents 3-4 from forming. Because transcription and translation are coupled in bacteria, the ribosome's position on the leader decides which hairpin forms. The ribosome stalls at the Trp codons when charged tRNA(Trp) is scarce. If no ribosome translates the leader at all, 1-2 and 3-4 form and transcription terminates.

The ara operon controls arabinose use. araBAD encodes ribulokinase (araB), L-arabinose isomerase (araA) and L-ribulose-5-phosphate 4-epimerase (araD). The regulator AraC is both a repressor and an activator. Without arabinose, an AraC dimer binds the araO2 and araI1 sites, which are far apart, and loops the DNA, which represses. With arabinose, AraC binds arabinose, changes conformation, releases araO2 and binds the adjacent sites araI1 and araI2, which activates transcription from the araBAD promoter. Activation also needs CAP-cAMP, so the ara operon, like lac, is subject to catabolite repression. AraC also represses its own gene, araC, by binding araO1.

Nitrogen fixation is the reduction of atmospheric N₂ to ammonia. Only certain prokaryotes (bacteria and archaea) carry it out. The enzyme is nitrogenase, which has two components: the Fe protein (dinitrogenase reductase, encoded by nifH) and the MoFe protein (dinitrogenase, encoded by nifD and nifK). The Fe protein, driven by ATP hydrolysis, passes electrons one at a time to the MoFe protein, whose iron-molybdenum cofactor (FeMo-co) binds and reduces N₂. Both components are irreversibly damaged by oxygen.

Protecting nitrogenase from oxygen. Leghemoglobin in the nodule (the globin is made by the plant) binds O₂ tightly, keeping free O₂ low while still supplying enough for bacteroid respiration. Heterocysts have thick walls and lack the oxygen-evolving photosystem II. Azotobacter respires rapidly. The nif genes (nifH, nifD, nifK, and regulators such as nifA) are switched off by high ammonia and by oxygen.

A pathogen causes disease through steps: adherence and colonisation, invasion, evasion of host defences, damage to tissue (often by toxins), and spread. Virulence factors are the molecules that make these steps possible: adhesins and pili, capsules (resist phagocytosis), secretion systems, iron-scavenging siderophores, enzymes (hyaluronidase, coagulase) and toxins. Many protein exotoxins are A-B toxins: the B part binds a receptor on the host cell and the A part is the active enzyme that enters the cell.

Antibiotics are selectively toxic: they hit targets that bacteria have and human cells lack or have in a different form (for example 70S versus 80S ribosomes). Bactericidal drugs kill bacteria; bacteriostatic drugs stop growth and rely on the host immune system to clear the infection. The minimum inhibitory concentration (MIC) is the lowest drug concentration that prevents visible growth in a defined culture after overnight incubation; the minimum bactericidal concentration (MBC) is the lowest that kills at least 99.9% of the cells.

Resistance spreads vertically by mutation and inheritance, and horizontally by conjugation (R plasmids), transformation and transduction, with transposons and integrons moving resistance genes and cassettes between replicons. Antibiotic use selects for resistant cells. Clavulanic acid, a β-lactamase inhibitor, is combined with amoxicillin to protect it.

A two-component system (TCS) is the usual way bacteria sense the environment. A sensor histidine kinase, usually a membrane protein, detects a signal and autophosphorylates a conserved histidine using ATP. The phosphoryl group is then transferred to a conserved aspartate on a cytoplasmic response regulator. Phosphorylation activates the regulator, which is often a transcription factor. The system resets when the phosphate is removed, either by the regulator's own slow dephosphorylation or by a phosphatase activity, often of the sensor itself.

Quorum sensing lets cells coordinate behaviour by measuring cell density through secreted signals called autoinducers. When the signal concentration crosses a threshold, a group of genes is switched on together. Targets include bioluminescence, virulence factors and biofilm formation.

A virus is an obligate intracellular particle whose genome is either DNA or RNA, not both, inside a protein shell called the capsid, built from capsomers. The capsid plus genome is the nucleocapsid. Some viruses also have a lipid envelope taken from a host membrane, carrying viral glycoproteins. Capsid symmetry is helical (tobacco mosaic virus), icosahedral (adenovirus, poliovirus) or complex (T4 phage, poxviruses). Viruses lack their own ribosomes and metabolism. Taxonomic classification uses the nature of the genome, capsid symmetry, presence of an envelope and size; the Baltimore scheme groups viruses by how they make mRNA.

Subviral agents. Viroids are small, circular, single-stranded RNA molecules with no protein coat and no protein-coding genes that infect plants (for example potato spindle tuber viroid). Prions are infectious proteins with no nucleic acid: a misfolded form of the prion protein (PrP) converts the normal form to the misfolded form, causing spongiform encephalopathies.

Bacteriophage cycles. In the lytic cycle a virulent phage adsorbs, injects its DNA, directs the synthesis of new phage, assembles and lyses the cell. In the lysogenic cycle a temperate phage such as lambda integrates as a prophage and replicates with the host. In lambda, the cI repressor maintains lysogeny. DNA damage triggers the SOS response: activated RecA (RecA*) stimulates self-cleavage of cI, and the phage enters the lytic cycle (induction).

One-step growth curve. After a synchronous infection, the eclipse period is the time during which no infectious phage can be recovered even from broken cells; the latent period lasts until the cells lyse and release phage. The burst size is the average number of progeny phage released per infected cell.

Transduction is gene transfer by phage. In generalised transduction (for example phage P1), random host DNA is packaged by mistake, so any gene can be transferred. In specialised transduction, a prophage that excises imprecisely carries only the host genes next to its integration site, as with lambda carrying gal or bio.