Prof.

Ivan Mitov 2008 - 2013

 Genetics is a science for HEREDITY and VARIABILITY оf
organisms, incl. microorganisms.

Genetics deals with the mechanisms of:

• hereditary transmission in the genera and

• variation of inherited characteristics of the species, resp. bacterial

species.
 MODERN OR MOLECULAR GENETICS BEGINS IN 1928 WITH AN FRED
GRIFFITH’s EXPERIMENTS ON Streptococcus pneumoniae
How has been discovered the chemical nature of the structure, encoding hereditary information?
S. pneumoniae
• important cause of morbidity and mortality in
humans;
• 1 bacterium cause lethal infection of mice;
• the capsule is the main factor of virulence;
• Non-encapsulated mutants are avirulent.

FRED GRIFFITH (1928): MIXTURE of killed by

heating encapsulated (virulent) bacteria and live
non-encapsulated (nonvirulent) bacteria kill the
mice.

Therefore some material from the killed cells

transferred the ability to the live non-
encapsulated bacteria to produce capsules.

Griffit called this phenomenon TRANSFORMATION.

That time the transforming principle was

unknown. The proteins have been suggested.
O. Avery, C.MacLeod and M. MacCarty continue to look for the
transforming principle and to answer the question "What is a
gene made of?“. In 1944 they succeeded to separate away from
all other cellular components the transforming substance – it
was composed only of DNA.

Therefore BACTERIA GAVE THE FIRST EVIDENCE OF THE

CHEMICAL NATURE OF GENES and marks the beginning of
the era of
• molecular genetics and
• contemporary biotechnologies.

.
. BACTERIAL GENOME
Structure
The bacterial genome consists of:
- bacterial chromosome and
- extrachromosomal genetic elements (nonessential)
- plasmids and
- temperate bacteriophages.

Chromosome

Plasmid

Bacteriophage

GENOTYPE and FENOTYPE

 The Chromosome
.In Escherichia coli and most bacteria:
-Single (haploid) circular molecule of double-stranded and superspiralized
DNA. Eukaryotic chromosomes are diploid.
Borrelia have linear chromosomes.
- 1,4 mm in length (i.e., nearly 1000 times the diameter of the cell)
- Approximately 5 000 000 base pairs (5000 kilobase [kb] pairs), or 4377 genes.

The smallest bacterial chromosomes (from mycoplasmas, chlamydiae and rickettsiae) are
approximately a fourth of this size. In comparison, humans have two copies of 23 chromosomes,
which represent 2.9 × 109 base pairs 990 mm in length.

.
 Size Gene Average base pairs Chromosome
Organism
(base pairs) number in the gene number

1 gene per 100,000

Homo sapiens 3.2 billion ~25,000 46
bases
1 gene per 100,000
Mouse 2.6 billion ~25,000 40
bases

Drosophila (fruit fly) 137 million 13,000 1 gene per 9,000 bases 8

Arabidopsis (plant) 100 million 25,000 1 gene per 4000 bases 10

Caenorhabditis
97 million 19,000 1 gene per 5000 bases 12
(roundworm)
Saccharomyces
cerevisiae 12.1 million 6000 1 gene per 2000 bases 32
(yeast)
Escherichia coli
4.6 million 4377 1 gene per 1400 bases 1
(bacteria)
H. influenzae
1.8 million 1700 1 gene per 1000 bases 1
(bacteria)
.Bacterial DNA (chromosome, plasmids) is SUPERCOILED

A collection of enzymes [DNA gyrase – (topoisomerase II),

topoisomerase I, etc.] is responsible for winding the DNA.

DNA gyrase twists the DNA about itself causing it to fold over at
every 200 base pairs. By repeating this process ≈ 20 000 times the
chromosome is organized into ≈ 50 supercoiled regions.

A fully supercoiled chromosome will be about 1 µm in diameter and is

small enough to fit inside a bacterium. Part of the chromosome is in
toroidal form (like spiral or selenoid).

The gyrase is a target for antibiotics:

novobiocin, nalidix acid and fluoroquinolones.

. Eucaryotic cromosomal DNA

.

Модел на ДНК-гираза и образуване на първичните усуквания.

На всеки 200 bp ДНК гиразата огъва ДНК веригата около себе си. Така в
хромозомата се образуват ≈ 20 000 усуквания. Механизмът е АТФ зависим.
Replication of DNA and bacterial chromosome
Both strands of DNA are:
3’ 5’ • complementary => A=T; C=G
• of plectonemic type – one strand must be cleaved to
unwind around the other;
• Each strand is polar – 3’-5’ ends
• Both strands are antiparalel.

3’ 5’
Replication of DNA
.The complementary double stranded structure of DNA
determine the semiconservatively way of replication:
daughter DNA strand are synthesized, using both strands of
the parental DNA as templates.

Replication of the bacterial genome is triggered by a

cascade of events linked to the growth rate of the cell.

Replication of bacterial DNA is initiated at a specific

sequence in the chromosome, called OriC. Several
enzymes and regulatory proteins take part:
• The helicase cleave and unwind the DNA at the
origin to expose single strands for synthesis of new
strands of DNA, forming the growing forks.
• The primase synthesizes primers to start the
process.
• The DNA-dependent DNA polymerases
synthesize a copy of the DNA, but only if there is a
primer sequence to add to and only in the 5' to 3'
direction: at growing forks and proceeds
bidirectionally. The leading strand is copied
continuously in the 5' to 3' direction, whereas the
other strand (the lagging strand) must be
synthesized as many pieces of DNA using RNA
primers (Okazaki's fragments). Then the pieces
are ligated together by the enzyme DNA ligase.

.
.

Replication of bacterial chromosome and plasmids. New DNA synthesis occurs at

growing forks and proceeds bidirectionally. It takes 40 minutes to complete one round
of replication. Cell division take about 25 minutes. Multiple growing forks are initiated
before complete septum formation, in order to supply enough copies of DNA for the
daughter cells. The daughter cells are "born pregnant."

Replication is complete when the two replication forks meet 180 degrees from the origin.
The process of DNA replication puts great torsional strain on the chromosomal circle of
DNA; this strain is relieved by topoisomerases (e.g., gyrase), which supercoil the DNA.
.

Model of F. Jacob, S. Brenner и F. Cusin for

replication of bacterial chromosome and the big
plasmids.
 Phage Types and Phage Infection
(Bacteriophages are bacterial viruses)

• Virulent phages infect sensitive bacteria,

replicate to large numbers and lyse the cell –
lytic infection.
• Lysogenic or temperate phages (E. coli λ-
phage, etc.) after infection integrate (prophage)
into the host genome without killing the host.
The bacterium multiplies, unaffected by the
infection. The offspring is lysogenized –
lysogenic infection.
Temperate phages remains lysogenic as
long as a repressor protein is synthesized
and prevents the phage from becoming
unintegrated and replicating independently of
the host chromosome. Activation of the
prophage may happened in some cells of the
lysogenic culture by damage of DNA by
radiation, chemical mutagens or by another
means. The integrated phage DNA is excised
from the bacterial chromosome and
replicates, thus turning to lytic cycle.
Some temperate DNA phages carry toxin
genes (e.g., corynephage beta carries the gene
for the diphtheria toxin), other genes change
bacterial antigens.
.
 Plasmids
Extrachromosomal nonessential genetic elements that replicate independently of the bacterial
chromosome (like chromosome they are replicons). Some plasmids, such as the E. coli F plasmid,
are episomes, which means that they can integrate into the host chromosome.
Double-stranded, circular DNA – 1,5 to 400 kB. (Borrelia burgdorferi, the causative agent of Lyme disease, and other
borreliae possess linear plasmids).

Plasmids carry genetic information, which may not be essential but can provide a selective
advantage to the bacteria. They may code:
Antibiotic resistance – conjugative R plasmids.
encoding the production of bacteriocins, toxins, virulence factors (Ent-, Hly-, Col-
plasmids) and enzymes (metabolic plasmids).

The multiple cloning site in the pGEM plasmid provides

The pBR322 plasmid is one of the different restriction enzyme cleavage sites for insertion of
plasmids used for cloning DNA: encodes DNA within the β-galactosidase gene (lacZ). The insert is
AmpR, TetR and an origin of replication flanked by bacteriophage promoters to allow directional
(ori). messenger RNA expression of the cloned sequence..
Large
. plasmids (20 to 120 kb), such as the
fertility factor F found in E. coli or the
R-plasmids which encode antibiotic
polyresistance, Ent-, Hly-, Col- and other
plasmids can mediate their own transfer
from one cell to another by a process called
conjugation. These conjugative plasmids
encode sex pili and all the necessary
factors for their transfer. They replicate
under strict control (one copy for each cell).

Small (cryptic) plasmids replicate in many

copies (without strict control) and distribute
irregularly between the 2 daughter cells.
The plasmids can be transferred into a
bacterial cell by transformation and
transduction also.

.
 Transposons (jumping genes)
Mobile genetic elements (Figure 5-8) that can transfer DNA from one position to another
in the genome or between different molecules of DNA (e.g., plasmid to plasmid or
plasmid to chromosome).
Transposons are present in prokaryotes and eukaryotes.
Insertion sequences (the simplest transposons) - 150 to 1500 bp, include gene
transposase (coding their own transfer), flanked with inverted repeats (15 to 40 bp at
their ends)

Complex transposons carry other genes, such as genes that provide resistance
against antibiotics. Tn-s are important tool for evolution of R-plasmids, differentiation of
the immunocompetent lymphocytes by gene rearrangement etc.
Integrons – Tn-s that carry several genes for resistance.

Transposons may insert into genes and inactivate those genes (insertional mutation).
A. IS10
tnp

Б. Tn10
tetR IS and Tn-s.
IS10 IS10 tnp - transposase
tet – gene for tetracycline
В. Tn3
bla – gene for β-
tnpA tnpR bla
lactamase
Some transposons have promotor regions. Dependent on their orientations they can
activate or inactivate the neighboring genes.

Transposons:
- mediate nonhomologous recombination.
- cause insertion mutations.
- are important tool in evolution of all species.
.
 GENE ORGANIZATION AND FUNCTION
The genes of the metabolic pathways in bacteria are organized in OPERONS. No
exons and introns.

Operons are groups of one or more structural genes expressed from a particular
promoter and ending at a transcriptional terminator.
Thus all the genes coding for the enzymes of a particular pathway can be coordinately
regulated.
Operons with many structural genes are polycistronic.

The operons are under control of protein repressors.

.
. Lac operon and inductive control of the operons in the catabolism

•The lactose operon is transcribed as a polycistronic messenger RNA (mRNA) from the promoter
(P) and translated into three proteins: β-galactosidase (Z), permease (Y), and acetylase (A). The
lac I gene encodes the repressor protein.
•In the absence of lactose the genes are not transcribed in mRNA, because the repressor
competes with the RNA polymerase at the operator site (O).
•In the presence of lactose the repressor, complexes with the inducer, change its
conformation and it can not block the operator. The lac operon is thus transcribed at a low level.

.
 Catabolic repression
The bacteria prefer easily assimilated substrates, such as glucose.

In the presence of glucose adenyl-cyclase is inhibited, the level of cAMP is low, no

formation of CAP-cAMP complex and the lac operon is repressed.

The CAP-cAMP complex enhances binding of the RNA polymerase to the promoter. E. coli in a poor
medium with lactose as the carbon source. Both the inducer and the CAP-cAMP complex are bound to the
promoter, which is fully 'turned on,' and a high level of lac mRNA is transcribed and translated.

.
Repressive control of operons in anabolic pathways

Regulation of the tryptophan (trp) operon.

A, The trp operon encodes the five enzymes necessary for tryptophan biosynthesis.
B, The conformation of the inactive repressor protein is changed after its binding by the
corepressor tryptophan. The resulting active repressor (R) binds to the operator (O),
blocking any transcription of the trp mRNA by the RNA polymerase. .
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