Genetic systems of prokaryotes Flashcards
(11 cards)
cycle
NB- if species not specified, assume E-coli.
Cycle through B (G1 equivalent), C (S, no G2), D (mitosis and cytokinesis) phases, with not B period in rapid growth. Circular chromosome represents minimum DNA per cell- rapidly dividing cells have >1 chromosome, generation time< replication time (overlapping replication rounds). DAPI stain-> chromosome= ~1/2 cell volume. Brownian motion-> randomly coiled structure ~10um diameter- further compaction to fit in cell while maintaining accessibility-> Nucleoid diameter ~1um.
Nucleoid organisation: supercoiling, topoisomerases
Supercoiling strands interlinked by geometry of double helix (twist= linking number (Lk) for relaxed molecule (no supercoiling). In eubacteria, -vely supercoiled/underwound, meaning strands of circular DNA duplex can’t separate completely by H-bonding, intertwining of backbones (twist/T). One interlink for each complete turn. In vivo, both chromosome+ plasmids have ~4% helix turns removed-> supercoiling/ writhe (W). Lk=T+W.
Topoisomerases I+ III-> ss break, II -> ds break, ATP hydrolysis (DNA gyrases (gyr) A+B), IV (parC/E) gyrase required for supercoiling, DNA rep and transc. TIV for decatenation of replication products. Mutation-> improper nucleoid segregation. In bacteria, -ve supercoiling by DNA gyrase (type 2 topoisomerase, consumes ATP). Type 1 (topoisomerase I) relaxes plasmid by ATP-independent supercoil removal- antagonistic action maintains constant supercoiling level. Also Topoisomerases III (type 1)+ IV (type 2) also involved.
Isolate nucleoids as membrane-free (MF)- 60%DNA, 30% RNA, 10% protein (RNAP); or membrane-attached (MA)- 56% protein, 17% DNA, 19% RNA. 8% phospholipid,+ 4 small, stable, +ve DNA binding+ bending proteins (nucleoid-associated proteins/NAPs), HNS, HU, FIS, IHF. Both unfold on heating/ DNase/RNase/protease.
HU, H-NS, MukBEF, HV
HU: compacts DNA @ low conc, form extended filaments @ high conc. Non-specific chromatin binding, purely architectural.
H-NS: bridging protein. Dimers have 2 outward-facing DNA binding domains extending from flexible liker, bridge distant DNA sites. Dimers assemble-> filaments (interact N/C termini of adjacent dimers)- scaffold like structure for nucleoid organisation (not fully understood yet). Silencer, in clusters (unlike non-specific HU).
MukBEF= SMC complex, organises chromosome by forming axial core (ATP hydrolysis), extrudes and constrains loops of 20-50kb. H-NS bridge adjacent loops.
HV has opposing effects: alpha-alpha form mediates expansion by summing weak+ transitory interactions of HU-DNA, non-specific binding through surface Lys. alpha-beta-> condensation by higher affinity looped/bent DNA.
Chromosome loop domains
Chromosome in loop domains of variable placement+ length, evidenced by EM, analogous to euk chromatin. Some topological isolation of chromosome sections. Some relaxed/ broken, some supercoiled:
Early speculation: some loop bases anchored by REP (repetitive, exogenic, palindromic- 290 regions x 1-12 tandem repeats copies x 40bp palindromic seq)+ BIME (bacterial interspersed mosaic element) seqs- palindromic, gyrase binding.
Irradiate w/ X-rays-> ~160 nicks per genome needed to relax 95%+/ superhelical tension, suggests ~120x 50kb loops per chromosome.
Recent studies suggest smaller (10kb) loops in E-coli, varying in position+ length. Experiment: express restriction endonuclease SwaI (cleaves 117 known sites in E-coli)-> relax loop in which cut, but adjacent loops unaffected. Microarray to assess cleavage effect on 300 genes (whose transcription sensitive to supercoiling)-> little response if >10kb from cut site. New cut sites introduced- no precise separation between gene+ cut site at which effect immediately apparent-> domain boundary positions vary cell to cell
Atomic force microscopy-> dynamic nucleoid structure. Open structure in exponential phase+ vice versa.
Gene regulation without a nuclear membrane
Transcription+ translation coupled. Some proteins exported+ membrane embedded w/ mRNA still attached to chromosome via RNAP-> chromosome transiently linked to membrane. May help maintain open nucleoid; drug inhibition of protein synthesis-> condensed nucleoid.
Attenuator-mediated ctrl genes involved in aa synth, exploits transc/transl coupling Trp operon leader mRNA can form mutually exclusive hairpins (BMB)- 3:4 (transcription terminator-) RNAP dissociate before reaching trp genes) or 2:3 (allow transcription), dependent on trp conc detected by translation of short polypeptide in leader region of trp mRNA. Leader seqs rich in cognate aa (trp here)- if lots of trp, ribosome continue to ORF end, sit on regions 1, 2-> 3:4 hairpin. Little trp-> ribosome pause-> 2:3 hairpin.
Prok chromosomes and plasmids
Most bacteria have 1 circular chromosome (as found in E coli, 1963). Linear chromosomes in species of Borrelia, Streptomyces. Multiple circular chromosomes in B. cepacia.
Combined core+ accessory genomes=pangenome.
Plasmids ctrl own replication, move horizontally-> allow bacterial evolution in network rather than as a tree+ rapid spread of antibiotic resistance. Increasingly viewed as independent, parasitic/ symbiotic.
Structure: Most studied plasmids= -vely supercoiled circles/dsDNA, E-coli. Largest only have 2-3% coding capacity/chr. Small typically have high copy #+ vice versa. Also can be linear- some contain terminal inverted repeats+5’ bound proteins to ensure complete replication (telomere problem); Borrelia has 2 strands of DNA duplex linked by single strand loop. Large plasmids (~200kb+) regarded as mini-chromosomes, e.g., R meliloti 1.4+1.7Mb “symbiotic megaplasmids” encoding essential housekeeping genes classified as chromosomes.
Plasmid phenotypes
- Resistance to antibiotics (e.g., chloramphenicol, macrolides),heavy metals (mercuric ions, Ni,Pb), toxic anions (arsenate/ite, borate), intercalating agents (acridines), radiation damage (UV/X ray), bacteriophage+ bacteriocins (+ plasmid-encoded restriction/modification systems)
- Metabolic properties- metabolism of simple and complex carbs/carbon/halogenated compounds, proteins, opines, N fixation; antibiotic and bacteriocin production
- Factors modifying bacterial lifestyle: Toxin production (->pathogenicity), colonisation antigens, haemolysin synthesis.
- Cryptic (unknown function) and plasmid-selfish (rep/maintenance/proliferation) genes
- Others: Gas vacuole formation, pock formation, rhizosphere protein, killing of host
Genes either plasmid-selfish to allow autonomous behaviour ot those that adapt host to circumstances that exist transiently/ limited by env- survive by natural selection when they increase host fitness (can also spread faster bot horizontally and vertically in these conditions)
Plasmid maintenance: low copy number
Low copy # plasmids use active partition (analogous to mitosis)- 2 families of Par (partition) cassettes well-studied- Plasmid F in P1 prophage+ plasmid R1. Each encodes 2 trans-acting proteins+ a cis site. Mutational studies-> mutation leads to plasmid instability/loss, while cloned Par can stabilise plasmid. Early models- “pre-pairing” (like pairing chromosomes in mitosis)- monomeric protein binds centromere-like site, dimerises to pair plasmids, requires molecular motor for separation after.
Par proteins from P1/F investigated: SopB/ParB bind sopC/parS-> partition complex.SopA/ParA (ATPases) bind complex-> ATPase activity on. ATP hydrolysis by SopA-SopB-sopC (or ParA-B-S) complex-> force to separate plasmid replication products. ParM-ParR-parC system in plasmid R1 EM showed plasmids paired in vitro by ParR binding parC, more efficient w/ ParM+ATP. Fluorescence microscopy of actively partitioned plasmids: new-born cell plasmids at mid-cell, move rapidly to ¼ and ¾ positions (middle of new cell after next division). Partition model in R1: ParM form filaments. ParM-ATP monomers enter complex @ parC, ATPase stimulated. ParM-ADP dissociates, allowing access to next ParM-ATP, polymerisation pushes plasmids apart.
Site-specific recombination assists active partitioning. P1 prophage lost <1/10k divisions, but in recombination-proficient hosts, some unstable P1 plasmids that are lost 1/100 divisions due to homologous recombination between P1 monomers-> dimer (can’t partition)- lack lox-cre region encoding site-specific recombination system. Cre recombinase mediates recombination between directly repeated lox sites in dimer, restores monomers. Chromosome dimers removed by Xer-dif site-specific recombination system in E-coli- dif close to terminus of chromosome, binds heterodimeric XerCD recombinase. Site-specific recombination doesn’t require extensive DNA homology. Interactions mediated by site-specific recombinases that bring together relevant seqs. Cre+ XerCD= Int family of recombinases. Reaction mech by sequential single strand changes. 1st exchange-> Holliday junction, then limited branch migration (may depend on recombinase/CG content/ physical coil strain), then 2nd strand exchange-> recombinant products.
Plasmid maintenance: high copy number
Multicopy/ high copy# plasmids: low probability of loss w/ random distribution. Dividing cell copy#-> rate of loss; relative growth rate of plasmid-containing+ plasmid-free cells-> rate of plasmid-free cell accumulation. Plasmids depress growth rate (add metabolic load). Evidence for strong clustering of plasmids in cells, #clusters«#plasmids-> Proposed that plasmid leaves cluster+ joins replication factory (mid-cell) to replicate. In absence of active partition, replication products distributed randomly. Then. Super-res fluorescence microscopy showed clustering less extensive than previously thought.
Multimerisation can cause instability for ColE1-like plasmids (multimers have lower copy# than monomers). Small#/multimers can have deleterious effect as concentrated in small proportion of population from which plasmid-free cells arise frequently. Concentration of dimers into ghettos due to replication advantage of dimers over monomers-> fast clonal accumulation in descendants of cell where dimer originally arose-> Dimer catastrophe. Detecting+ eradicating multimers vital to multicopy plasmids due to copy#ctrl blind spot.
Multimer resolution systems: natural multicopy plasmids carry recombination sites- host proteins convert multi->monomers. Cer-Xer of ColE1= best studied multimer resolution system- plasmid has 240bp cer site, where host recombinases XerC+D+ accessory proteins ArgR+PepA mediate site-specific recombination, under strict topological constraint (reverse reaction strongly supressed). 2 proposed models:
* Oxford model: recombining sites inter-wrap-> active recombination complex- for supercoiled substrates, almost impossible is recombining sites on different plasmids, but not if on same one.
* Cambridge model: sites brought together by weak bridge of hexameric ArgR+ PepA- can’t hold them together long enough alone for recombination, but when cer sites on same molecule, association reinforced by spring clip effect of DNA supercoiling (absent in monomers)
NB: in industry, payoff between getting plasmids to produce desired protein+ that process’ cost to bacteria, which increases host generation time. If generation time w/ vs w/out plasmid 1:1, no fitness cost. Plasmid washout relatively fast (15-20 gens) w/out selection.
Conjugative plasmids and mechanism for conjugation
Conjugative plasmids (F, R factors) allow horizontal gene transfer- nucleic acid doesn’t leave protective cell env (away from extracellular nucleases). Multiple genetically distinct, non-interacting conjugation systems found. in Gram-negative bacteria, plasmids direct synthesis of extracellular pilus-> recognition of recipient cell, establishment of cell-cell contact. Originally thought F hosts v. limited, later found could transfer E-coli-> yeast/mammalian cells.
Conjugation mech: >30 genes required for conjugation, cluster in 33kb tra region (~1/3 of plasmid)- at least 14 genes for F-pilus formation (8nm hollow cylinder w/2nm axial hole). Initial contact-> pilus shrinks (current models suggest pilus disassembly crucial to establish cell surface contact, format of DNA transport pore). Single strand nick @ plasmid origin of transfer, nicked strand transferred to recipient. Non-transferred strand copied. Conjugation involves extra replication round, provides mechanism for plasmid to proliferate faster than host.
F factor encodes mobile elements- insertion seqs (IS) or transposons (Tn), classed on basis of genome organisation and mech of transposition.
Insertion sequences and Tn3 family
Insertion seqs: 750-1600bp, have transposase gene flanked by terminal inverted repeat seqs (15-50bp)- binding seqs for transposase. Cut+ paste mech- transposase-> ds cuts @ inverted repeat ends+ staggered cut in target DNA, ligates IS into new location, linearising donor replicon (that may be degraded or repaired).
Composite transposon= when 2 IS elements flank gene (e.g., for antibiotic resistance). If transposase acts on outer pair, whole composite transposon moves, but individuals ISs also retain independence. Easily formed, giving all genes inproks potential to move genome to genome+ conjugate to other species.
Tn3 family: more complex structure than IS. Flanked by short inverted repeats, encode transposase and resolvase+ resistance to 1+ antibiotics. Transposase-> concerted replication+ transposition, generating cointegrate structure where donor+ target replicons joined by copies of transposon. Resolvase= site-specific recombinase, acts at res sites in each transposon copy in cointegrate, separating donor+ target molecules. Clinical significance- novel antibiotic resistances arise in these.
