E.

coli systems and recombination: Determinants of

diversity: Overall aims ML
 Ten lectures with Key topics.
 Homologous recombination and DNA repair
 Role of methylation and repair.
 Role of Plasmids; control of replication, transfer and stability.
 Illegitimate recombination: transposons and integrons
 Regulation of DNA transposition.
 You should:
 Have a basic grounding for further reading and other systems
covered in the course (e.g pathogens).
 Be able to critically read key papers in the area.
 Critically assess the development of ideas to date.

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 References

 http://www.blackwellpublishing.com/trun/
http://www.blackwellpublishing.com/trun/artwork/Animations/Recombination/recombination.html

Fundamental Bacterial Genetics

Janine Trempy, Nancy Trun

•Paperback 300 pages (2003)

•Publisher: Blackwell Science (UK);
ISBN: 0632044489

Molecular Genetics of Bacteria Dale and Park

.
•Paperback 352 pages (15
January, 2004)
•Publisher: John Wiley and
Sons Ltd; ISBN: 047085085X

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Role of Plasmids; control of replication, transfer and
stability.
 Review of DNA replication
 Errors in replication and their reversal
 Plasmid replication initiation and its control
 ColE1 and R1 as examples
 Plasmid segregation, recombination and stability
 Control of plasmid transfer
 Mobilisation functions
 Plasmid structure and evolution
 Antibiotic resistance and catabolic functions
 You should be able to discuss the importance of negative control
of plasmid functions and their critical importance in the evolution
of diverse microbial genomes
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
 Overview of DNA replication
5’
3’

3’

5’

5’
PolIII Prepriming Pol I
3’
proteins
Helicase Ligase
Ssb Uracil
glycosylase
Primer
DNA
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
 Proofreading DNA polymerase

Removal of 3’ - 5’ digestion by polymerase

mismatch

AG or CT Mismatch

Polymerase
Polymerisation in 5’ - 3’ direction

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Initiation of Chromosome replication
Hu ATP DnaA
3 x 13 mers 4 x 9 mers

oriC

Dna B,C ATP

Prepriming complex
Replication

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Plasmid replication and control
Generally a plasmid only has to reveal an origin of replication within the cell and
“hijack” the cell’s replication mechanism. But it must not over replicate too
many copies and damage the cell’s functions.

THREE initiation strategies are known.

1. Mimic chromosome origin as on previous slide e.g F, R1, phage P1 and 

2. Plasmid encoded Rep protein initiator - ss Nick - ss template for synthesis
e.g. pT181 in Staph. aureus (also in other Gm+ves) (Summers 1986)
3. Primer RNA initially synthesised from constitutive promoter. E.g ColE1

ColE1 as an example : extensively studied

Different initiation mechanism to chromosome
No plasmid encoded Rep protein needed to initiate replication

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 ColE1 replication

Small Plasmid - 4.3 Kb

replication UNIDIRECTIONAL
Approx’ 15 copies per cell
Encodes colicin E1 and…
ONE protein required in replication
oriV

INITIATION OF SYNTHESIS

oriV start
RNA II primer formed first
Starts 555 nt BEFORE oriV
Synthesis catalysed by HOST DNA dependent RNA Polymerase
Normally used in gene transcription
HOST DNA replication mechanisms then take over
MUST be a mechanism for negative regulation also.

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 ColE1 replication cont……..

oriV L or Light strand

3’ 5’
5’ 3’
H or Heavy strand
Poly U or Poly G tails / CsCl density grad’ centrifugation
New L Strand synthesised first
ONLY 580 bps needed for plasmid to replicate in host cell
RNAI RNA Polymerase
-445
RNAII starts at -555 on H strand
- 555
RNAII

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 ColE1 replication cont……..
-445 Transcription beyond oriV
Hybridisation at/near oriV
- 555
3’

-445 RNAaseH cleaves at oriV

2/3 bp accuracy
- 555
3’

3’
DNA PolI begins synthesis
-445
on primer RNA

ONLY 580 bps needed and ONLY 13 after oriV

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 ColE1 replication cont…Chain elongation..

New H - strand synthesis

Begins after initial L-strand formation
Discontinuous in 5’ to 3’ direction as Okazaki fragments
Fragments about 1000 bps long initiate on short RNA primers
DnaB forms secondary structure in ss DNA
Allows initiation by primase DnaG
DNA PolI extends primer

Continued L - strand synthesis

DNA PolI continues for about 500 bps after initiation
DNA PolII and primosome then take over
L- strand extended as leading strand synthesis

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Control of ColE1 replication
Replication control in relation to host cells growth rate?
E. coli may replicate as slowly as 12h in gut and up to 40min in lab’!
Efficient ColE1 maintenance.
Negative control of replication initiation - low copy number

TWO products involved

Mutational studies on ColE1

RNA I and Rom (Rop) protein

RNA I is ANTISENSE message

Binds RNA II primer to prevent RNAase H processing
Binding enhanced by Rom protein
Cloning vectors (based on pMB1 related to ColE1) have no rom gene
Copies may exceed 200-300 per cell !

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Control of ColE1 replication cont…...
ANTISENSE RNA I binding to complementary RNA II
NOT a simple base pairing mechanism.

SECONDARY STRUCTURE is important

Mutational studies reveal regions in the RNA I molecule necessary for control
G
G
U
Rom A
G

G
A

5’ Necessary for antisense

3’
RNA I secondary structure binding

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Summary of ColE1 replication control

oriV

+600 +400 -500

5’
3’

RNAase cut
3’

Rop/Rom
Dimer 5’ 3’

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Large conjugative plasmid replication control
•ColE1 is a small NON-CONJUGATIVE plasmid
•Many others are much larger and CONJUGATIVE or TRANSMISSIBLE
•(NOTE ColE1 can be transferred by mobilisation functions)
•Larger plasmids often present as 1 or 2 copies per cell
•There may be two possible ori sites
•Replication is often bidirectional
•The replication of closely related plasmids R1, R100 and R6-5 studied in detail
•THESE HOWEVER REPLICATE UNIDIRECTIONALLY
•R1 and R100 are approx’ 90Kb in size
•All but 2 Kb can be removed without blocking replication
•Must contain the oriV site
•Regulation differs from ColE1 regulation

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Regulation of R1 replication
copA and copB mutations lead to x 10 increase in copy number
Tap ia a transcriptional activator protein
repA mutants fail to replicate RepA doubles for host Pol1

RNA I antisense
5’ 3’
copB copA tap repA oriV
3’ 5’
RepA initiates
RNA II replication

Tap

Repressor
Shine-Delgarno repA

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Regulation of other plasmid replicons
F plasmid
Similar to R1 but does not depend upon RNA/RNA interactions

 - Phage replication
Action of Cro O and P proteins CroO is an autorepressor

P1 - prophage and some other plasmids

ori (incC) region “HANDCUFFED” to incA region after replication
Multiple 19 mer repeats
RepA binding involved
Works in cis and trans

RepA Also works in trans

ori incA HANDCUFFED linking TWO P1 DNA
NO repA transcript molecules
repA
repA

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Plasmid segregation and stability

INCOMPATIBILITY
Plasmids with similar replication control are unstable
Belong to the same incompatibility (Inc) grouping
Or Incompatibility may be related to similar partitioning functions
See below

RANDOM PARTITIONING
High copy number plasmids show random partitioning
Recombination to multimers
Multimers unstable
Must be resolved

ACTIVE PARTITIONING
Proteins make sure that copies of plasmids are segregated on cell division
SopA and SopB in F plasmid
ParA and ParB in P1 phage

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Resolution of multimeric plasmids

High copy number plasmids should be stable

However vector plasmids such as pUC, pBR322 etc.. are very unstable
particularly in rec+ hosts or sbcA mutants
MULTIMERS formed easily and not resolved

However ColE1 is stable

Why?
It has a 240bp sequence called cer that is needed for stability
Similar to resolution site on Tn3 family of transposons
Acted on in trans by XerB and XerC proteins
Host encoded
Usually act on dif sequence to resolve chromosome after replication

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Resolution of multimeric plasmids

240bp cer sequence

C
+ D

Xer C and Xer D

Chromosomally
encoded
C
C
D Form Dimer D

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Active partitioning of plasmids
sopA/parA
pSC101 par region
375 bp region
Binding site for DNA gyrase

F1 and P1 partitioning
sopA and B parA and B

ParB/SopB
ParB/SopB for a dimer
Bind to sopA / sopB
Associate with cell membrane
and accurately segregate

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Regulation of plasmid transfer

Will consider F and related R plasmids

Then mobilisation of colE1

Chromosomal integration
IS3, IS2, 
33 Kb transfer
Very complex transfer system
genes region
t.ra genes transcribed from
4 promoters in traJ region
F Plasmid
94.5 Kb

oriT oriV and rep region

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.

 Regulation of plasmid transfer cont….
Antisense RNA involved

F System
finP
tra genes traJ
IS3

R100 System finO BLOCKED

finP cannot bind

finP
tra genes traJ finO

Binding and finO finO

repression
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
 Plasmid Mobilisation
Many plasmids NOT conjugative
But CAN be MOBILISED by transfer functions in other plasmids
ColE1 a good example

mob and bom functions involved

mob genes on plasmid substitute for tra genes
Act on bom sequence as for oriT

NO F plasmid present F plasmid present

Bom site nicked by
bom After nicking of bom site
Mob proteins
Transfer substrate is present
but NO transfer
F transfer functions transfer
the col plasmid

mob

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.