29-04-2018

2017-18
Genetica Molecular

Genética de Bactérias e
seus Vírus-P1

Circular
genetic
map of E.
coli

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O que têm que saber:

• Organização do genoma da bactéria
• Estrutura e função de plasmídios
• Mecanismos de recombinação:
– Crossing over
– Sequências repetitivas directas e invertidas
– Transposões
– Inserção e excisão de sequências
• O que é um bacteriofago

Alguns Factos:

- Muitas das bactérias isoladas em hospitais

apresentam múltiplas resistências a antibióticos.
- Essas resistências não provêm em geral de
mutações cromossómicas.
- São adquiridas a partir de elementos móveis de
DNA, por vezes associadas em grupos.

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Plasmids

• Plasmids are circular DNA molecules that replicate

independently of the bacterial chromosome

• They are mobile sequences and can be transferred

between bacterial individuals and among species.

• Plasmids often carry antibiotic resistance genes

• Plasmids are used in genetic engineering as gene

transfer vectors

Bacterial Genetics

Three principal types of bacterial mutants use in

bacterial genetics:
• Antibiotic-resistant mutants are able to grow in the
presence of an antibiotic, because, in the presence of
antibiotics
• Nutritional mutants are unable to synthesize an
essential nutrient and thus cannot grow unless the
required nutrient is supplied in the medium. Such a
mutant bacterium is said to be an auxotroph
• Carbon-source mutants cannot utilize particular
substances as sources of energy or carbon atoms

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Transposable Elements in bacteria

• Transposable elements are DNA sequences that can
jump from one position to another or from one DNA
molecule to another.
• Bacteria contain a wide variety of transposable
elements
• They can allow homologous recombination between
different DNA molecules
• The smallest and simplest are insertion sequences, or
IS elements, which are 1–3 kb in length and encode
the transposase protein required for transposition
and one or more additional proteins that regulate the
rate of transposition

Transposable Elements

• Other transposable elements in bacteria contain one

or more genes unrelated to transposition that can be
mobilized along with the transposable element

• Transposons can insert into plasmids which can be

transferred to recipient cells by conjugation

• Transposable elements are flanked by inverted

repeats and can contain multiple antibiotic
resistance genes

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Mechanisms of transposition

Transposable Elements
• Integrons are DNA elements that encode a site-specific
recombinase as well as a recognition region that allows other
sequences with similar recognition regions to be incorporated
into the integron by recombination.
• The elements that integrons acquire are known as cassettes
• Integron may acquire multiple-antibiotic-resistance cassettes,
which results in the plasmid resistant to a large number of
completely unrelated antibiotics
• Bacteria with resistance to multiple antibiotics are an increasing
problem in public health (Ex: in Tuberculose)

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Site-specific recombinase

Integron
sequentially
captures
cassettes

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Cassette excision from integron

Cointegrate
formed
between two
plasmids (1)

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Cointegrate
formed
between two
plasmids (2)

Mechanisms allowing introduction of

exogenous DNA into bacterias

• Transformation (DNA + bacteria)

• Conjugation (bacteria + bacteria)
• Transduction (bacteria + virus)

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Bacterial Transformation
• The process of genetic alteration by pure DNA is called
transformation
• Recipient cells acquire genes from DNA outside the cell
• DNA is taken up by cell and often recombines with
genes on bacterial chromosome
• Bacterial transformation showed that DNA is the
genetic material
• Transformation may alter phenotype of recipient cells
– Ex: Plasmids or linear DNA

Detection of recombinant plasmids through insertional

inactivation of a fragment of the lacZ gene from E. coli

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Transformed bacterial colonies

Cotransformation of Linked Genes

• Genes located close together are often transferred as a

unit to recipient cell = cotransformation

• Cotransformation of two genes at a frequency

substantially greater than the product of the single-gene
transformation frequencies implies that the two genes
are close together in the bacterial chromosome.

• Genes that are far apart are less likely to be transferred

together

• Cotransformation is used to map gene order

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Cotransformation of Linked Genes

Cotransformation of Linked Genes

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Conjugation

• In conjugation, also known as bacterial

“mating”, DNA transfer is unidirectional

• F factor can integrate into the bacterial

chromosome via genetic exchange between IS
elements present in F and homologous copy
located anywhere in bacterial chromosome

F factor and Conjugation

• F (fertility) factor is a conjugative plasmid transferred

from cell to cell by conjugation

• F factor is an episome = genetic element that can insert

into chromosome or replicate as circular plasmid

• The F plasmid is a low-copy-number plasmid ~100 kb in

length, and is present in 1–2 copies per cell

• It replicates once per cell cycle and segregates to both

daughter cells in cell division

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F factor and Conjugation

• Conjugation is a process in which DNA is transferred

from a bacterial donor cell to a recipient cell by cell-to-
cell contact

• Cells that contain the F plasmid are donors and are

designated the F+

• Cells lacking F are recipients and are designated the F-

• The transfer is mediated by a tube-like structure called

a pilus, formed between the cells, through which the
plasmid DNA passes

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Conjugation and HFr Cells

• The F plasmid, being a episome, can integrate into

the bacterial genome

• Cells with the F plasmid integrated into the bacterial

chromosome are known as Hfr cells

• Hfr = high frequency of recombination

• In an Hfr cell, the bacterial chromosome remains

circular, though enlarged ~ 2 percent by the
integrated F-factor DNA

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Conjugation and HFr Cells

• When an Hfr cell undergoes conjugation, the

process of transfer of the F factor is initiated in the
same manner as in an F+ cell

• However, because the F factor is part of the

bacterial chromosome, transfer from an Hfr cell also
includes DNA from the chromosome

Conjugation and HFr Cells

• Transfer begins within an integrated F factor and

proceeds in one direction
• A part of F is the first DNA transferred, chromosomal
genes are transferred next, and the remaining part of F
would be the last to transfer (highly unlikely to occur)
• The conjugating cells usually break apart long before the
entire bacterial chromosome is transferred, and the final
segment of F is almost never transferred

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Integration of F by recombination

Conjugation of an HFr cell with a F- cell

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Conjugation of an HFr cell with a F- cell

Conjugation of an HFr cell with a F- cell

• The conjugating cells usually break apart long before the
entire bacterial chromosome is transferred, and the final
segment of F is almost never transferred

• The recipient cell remains F-

• A fragment from the bacterial chromosome transferred

from the HFr cells is integrated by homologous
recombination into the genome of the F- cell.

• The F- cell acquires new DNA and eventually new

characteristics

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