Microbiology

rt 1 · The Bacterial

Genome

CHAPTER DIVIDER

The Bacterial Chromosome

• Single, circular, double-stranded DNA molecule — no histones,

no nuclear membrane

• Occupies the nucleoid region of the cytoplasm

• E. coli: 4.6 Mbp, ~4 300 genes — in a cell 1000× smaller than a

human cell

• Exceptions: Vibrio cholerae (two chromosomes); Borrelia (linear

chromosome)

Essential vs. Accessory Genome

Essential Genome (Chromosome)

Replication, transcription, translation, core metabolism — the genes every cell needs to survive

Accessory Genome (Plasmids, Transposons, Integrons, Prophages)

Resistance genes, toxins, adhesins — what makes one strain more dangerous than another

The accessory genome is the part that moves — between cells, species, and hospitals.

Plasmids

• Replicate independently of the chromosome; 1–5% its size; mostly circular

dsDNA

• Copy number: 1 to >100 per cell; supercoiled in vivo; episome if integrated

• R (resistance) plasmids – spread antibiotic resistance by cell-to-cell contact;

multiple resistance genes on one molecule

• Virulence plasmids – encode adhesins, toxins, enzymes (e.g. E. coli

haemolysin, enterotoxin)

• Bacteriocin plasmids – encode narrow-spectrum proteins that kill related

bacteria

The R100 Plasmid – Multidrug Resistance in One

Molecule

• Five resistance genes on one 94.3 kbp plasmid — select

for one, select for all

• Carries its own transfer machinery (tra, oriT) — self-

transmissible among Escherichia, Klebsiella, Proteus,

Shigella, Salmonella

• Mercury resistance beside antibiotic resistance — heavy-

metal exposure co-selects for AMR

• Genes: tet (tetracycline), cat (chloramphenicol), sul

(sulfonamide), str (streptomycin), mer (mercury),

IS1/IS10/Tn10

Mobile Genetic Elements

Insertion Sequence (IS)

150–1500 bp; encodes only transposase; flanked by inverted repeats — inactivates genes on insertion

Transposon (Tn)

Resistance/toxin genes flanked by IS elements — jumps between plasmid and chromosome; Tn3 carries β-lactamase

Integron

Capture system with integrase + promoter — assembles resistance cassettes in blocks (Class 1 = engine of MDR)

ICE

Transposon + transfer machinery — moves between cells without a plasmid; carries vanB, tetM

Prophage

Integrated phage genome — lysogenic conversion (diphtheria, cholera, Shiga toxins)

Part 2 · Replication and

Expression

CHAPTER DIVIDER

DNA Replication in Bacteria

• Single origin (oriC); bidirectional

replication forks form a theta (θ)

intermediate

• E. coli: 4.6 Mbp replicated in ~40 min

at 1000 nt/sec/fork; error rate 1 in 10⁹

after proofreading

• Fast-growing cells start a second

round before the first finishes —

enabling 20-min generation time

The Replication Fork – Key Enzymes

Helicase

Unwinds parental

strands by breaking H-

bonds

Primase

Lays down RNA primer

(polymerase cannot

start de novo)

DNA Polymerase

Extends 5′→3′ with

proofreading; leading

strand continuous,

lagging strand in

Okazaki fragments

DNA Ligase

Joins Okazaki fragments (reappears

in cloning, Part 6)

Topoisomerase / DNA

Gyrase

Relieves supercoiling ahead of fork;

target of fluoroquinolones, which inhibits the function of DNA replication

/The Central Dogma = A Map of Drug Targets
DNA Gyrase
Fluoroquinolones → resistance:
gyrA/parC mutations, qnr plasmids
Folate Synthesis
Sulfonamides, trimethoprim →
resistance: sul, dfr genes (often on
integrons)
RNA Polymerase β
Rifampicin → resistance: rpoB point
mutation
30S Ribosome
Aminoglycosides, tetracyclines → resistance: modifying
enzymes, tet efflux
50S Ribosome
Macrolides, clindamycin, chloramphenicol, linezolid →
resistance: erm methylases, cfr, 23S rRNA mutations
Read this table again in Week 8 — same table, two directions.

Why Regulate Gene Expression?
Making an unnecessary enzyme wastes amino acids, ATP, and ribosome time. In a
cell dividing every 20 minutes, that metabolic cost decides who outgrows whom.
Rule: catabolic → inducible; anabolic → repressible
Inducible Operon
Normally off; switched on when substrate appears →
catabolic pathways (e.g. lac)
Repressible Operon
Normally on; switched off when end product accumulates →
anabolic pathways (e.g. trp)

Operon Anatomy
01
Promoter
Where RNA polymerase binds to begin transcription
02
Operator
The controlling site; a molecular traffic light (go/stop)
03
Regulatory Gene
Encodes the repressor protein
04
Structural Genes
Encode the enzymes of the pathway

The lac Operon – Inducible
When lactose is the sole carbon source, three genes are induced ~1000-fold simultaneously:
lacZ
β-galactosidase:
splits lactose →
glucose + galactose;
also makes
allolactose (the
actual inducer)
lacY
Lactose permease:
transports lactose
into the cell
lacA
Transacetylase:
function still poorly
understood

Negative Control – The Repressor
No Lactose
Active repressor binds operator → blocks
RNA polymerase → no transcription
Lactose Present
Allolactose binds repressor → repressor
changes shape → cannot bind operator →
transcription proceeds
Allolactose is an anti-repressor, NOT an
activator — it removes the block, it does not switch
the operon on directly

Positive Control – CAP, cAMP and Catabolite Repression
Removing the repressor is not enough — the lac promoter is weak.
• CAP–cAMP complex binds upstream of promoter, bends DNA, helps RNA
polymerase bind (positive control)
• cAMP is inversely proportional to glucose — glucose drives cAMP down
• Glucose +, Lactose +: repressor removed, CAP–cAMP low → very low
transcription
• Glucose –, Lactose +: repressor removed, CAP–cAMP high → maximal
transcription

The trp Operon – Repressible
• Low tryptophan: Repressor inactive → cannot bind
operator → enzymes transcribed
• Tryptophan present: Trp acts as co-repressor → binds
inactive repressor → activates it → blocks RNA
polymerase → no transcription
• Logic: stop making enzymes for a product you already
have
• Key contrast: in lac, small molecule inactivates
repressor; in trp, small molecule activates it — same
architecture, opposite switch