This document discusses the organization and segregation of bacterial chromosomes using Escherichia coli as an example. It finds that: (1) two markers on the same chromosome arm colocalize in the same cell half, while markers on opposite arms are in opposite cell halves, (2) duplicated chromosome arms are usually oriented in a tandem repeat configuration, and (3) sister cells are not usually identical after cell division due to rearrangement of chromosome arms.

1. Asha Ann Philip
11/16/2017

2. • Escherichia coli is a Gram negative, facultative anaerobic, rod-shaped bacteria.
• It is a commensal that is found inhabiting the lower intestine of warm blooded
animals; a small proportion of E. coli strains are pathogenic.
Escherichia coli (E.coli)

3.  Chromosomes are the structures that contain
the genetic material
 They are complexes of DNA and proteins
 In bacteria, it is typically a single circular
chromosome
 In eukaryotes, it refers to one complete set of
nuclear chromosomes
Chromosomes

4. • Bacterial chromosomal DNA is usually a circular molecule that is a few
million nucleotides in length.
◦ Escherichia coli ~ 4.6 million base pairs
◦ Haemophilus influenzae ~ 1.8 million base pairs
• A typical bacterial chromosome contains a few thousand different genes
◦ Structural gene sequences (encoding proteins) account for the majority of
bacterial DNA

5. Nucleoid
The nucleoid (meaning nucleus-like) is an irregularly shaped region within
the cell of a prokaryote that contains all or most of the genetic material,
called genophore.
In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a
nuclear membrane

6. Prokaryotic chromosome is consists of single circular, double-
stranded DNA molecule.
∗ It has no histone protein.
∗ Chromosomes are stored in nucleoid in which DNA is binding
to some proteins.
∗ Contain only one copy of the gene (hapolid).
∗ Non essential genes are stored outside of chromosome in
plasmid.
 The first complete DNA sequence of an E. coli genome (laboratory strain K-12 derivative
MG1655) was published in 1997.
 Greater gene density is due to a combination of factors:
∗ The genome of prokaryotes is often significantly larger
than the cell it self. How it is possible to keep the
information?
(1) bacterial genes have no introns
(2) neighboring genes are very close together throughout the genome;
i.e., there are hardly any big regions of non-coding DNA between genes.

7. A few hundred
nucleotides in length
These play roles in DNA folding,
DNA replication, and gene
expression
 The chromosomal DNA must be
compacted about a 1000-fold
(formation of loop domains).
 E. coli has 50-100 with 40,000 to
80,000 bp of DNA in each.

8. Replichores

9. Big Question
Conclusion: DNA replication and segregation results in considerable disorder in the
nascent nucleoids, some unknown mechanism rearranges the chromosome arms in
dividing cells with respect to the cell quarters, so that newborn cells have an orderly
separation of the arms.
How does the orderly arrangement occur in the chromosome?

10. • How can we understand the chromosome duplication and
segregation dynamics?
Fluorescence in fixed cells by in situ hybridization (FISH) Niki and
Hiraga, 1998).
By using fluorescent techniques such as the GFP-ParB/parS system
(Li et al., 2002)
GFP-LacI/operator system (Gordon et al., 1997; Lau et al., 2003)
Physical positions of specific proteins or DNA sequences in the cell.
Using these techniques, it is possible to determine the movement of
individual parts of the chromosome as they are replicated and
segregated and detect the position of discrete DNA loci inside the living
cell.

11. Approach: Plasmid partition system

12. • Plasmid :pFHC2973 (the P1-parB gene and the pMT1-parB gene fused
to the genes for the fluorescent proteins CFP and yGFP)

13. Cyan P1
parS site
Green
pMT1 parS
site

14. Results
Detection of two loci simultaneously using two plasmid parS sites of
different specificity
 P1 plasmid partition site P1parS and CFP-D30ParB (Dabrazhynetskaya et al., 2005 )
 P1-like partition system of the pMT1parS and yGFP-D23ParB (Nielsen et al., 2006)

15. Progressive segregation of the Escherichia coli chromosome
Molecular Microbiology
Volume 61, Issue 2, pages 383-393, 12 JUN 2006 DOI: 10.1111/j.1365-2958.2006.05245.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2006.05245.x/full#f1
(Li et al., 2002).

16. Fig B shows cells of the FH2973 strain (P1parS at
54′ and pMT1parS at 22′).
Fig A shows cells of the FH3518 strain
(P1parS at 15′ and pMT1parS at 22′)

17.  Two markers on the same chromosome arm colocalize in the same cell.
Markers on opposite chromosome arms are in opposite cell halves
Same side 1c1g
Opposite sides 1c1g
FH3518 strain FH2973 strain

18. Two markers that are on the same chromosome arm are in the same
cell half, whereas two markers on opposite arms of the chromosome
are in opposite cell halves.
Interpretation

19. Duplicated loci from opposite arms colocalize temporarily on the
inside of the cell quarters
The 2c2g subpopulation appears to represent a stage in which the chromosome is disorganized, and will
later be reorganized so that he chromosome arm will be in opposite cell halves in the next generation

20.  Subset of the smaller (youngest) cells in the 2c2g with a subset of the septated ones that were
about to divide
• colocalization of the 22′ and 54′ loci within the cell quarter positions appears to
be an intermediate step.
• Nucleoid will reorganize so that the loci will span the cell quarters and will be on
opposite sides of the cell centre after cell division.
Small 2c2g (n=83)
Dividing cells (n=83)

21. The spatial organization of the chromosome arms into opposite halves of the
cell involves about half of the chromosome, and presumably more.
Same side 1c1g
Opposite sides 1c1g

22. Duplicated chromosome arms are usually oriented in
a tandem repeat configuration
Two different relative orientations of the nucleoids in dividing cells
1. Symmetrically arranged, in which case the distribution of intermediate
markers would be symmetrical about the cell centre.
2. Arranged in tandem, in which case one copy close to the pole and its sister
locus just on the other side of the middle
The arranging of the chromosome arms results in a parallel
configuration of the two nucleoids prior to cell division.

23. Sister cells are not usually identical: the majority has
opposite nucleoid organizations
The arm carrying the 14′ marker would be adjacent to the new
pole in one cell of a newborn pair, and adjacent to the old pole
in the other.
33′
14′

24. Chromosome rearrangement model.
Sister loci cohesion

25. Replication

26. SUMMARY
Both daughter cells produced by binary fission in bacteria are more or less
identical to the parent.
The two nucleoids are oriented identically and that newborn sisters both have a particular
arm of the chromosome adjacent to the new pole.
The two markers that are on the same chromosome arm are in the
same cell half, whereas two markers on opposite arms of the
chromosome are in opposite cell halves.