Genetics - Chapter 6 - Bacterial Genetics

Bacteria have genes for traits

Bacterial traits include the ability to make molecules, use nutrients, detect “things” in the environment, produce toxins or other substances

Bacteria replicate their DNA and divide the products into the two daughter cells (vertical gene transfer); cell division

• Bacteria do not have mitotic or meiotic cell cycles

• Chromosomes do not condense but are replicated and apportioned to the two daughter cells

Horizontal gene transfer (HGT)

• Any process in which an organism incorporates genetic material from another organism without being the offspring of that organism

• Also (rarely) called lateral gene transfer (LGT)

Bacteria

Microbes: all bacteria are microbes BUT not all microbes are bacteria

Prokaryotic (NOT eukaryotic): no internal membrane compartments

Benefits of using microorganisms in research

• Bacteria and bacteriophages are essential in genetic study

• Bacteria and virus research is valuable

  • Exhibit extremely short reproductive cycles

  • Can be studied in pure or mixed cultures

  • Many bacterially and virally-caused diseases still have no cure or effective treatments available

  • Simplest window into the intricacies of the cell

All bacteria have:

• Nucleoid, an area of the cytoplasm that contains DNA

• Cytoplasm, filled with ribosomes

• Cell membrane

• Cell wall

Some bacteria have:

• Flagella, one or more

• Pili (s. pilus), important for adhesion to host cells

• Capsule, polysaccharide; provides resistance to phagocytosis

Bacteria are diverse:

• They have a wide variety of cellular morphologies

• They perform the greatest range of biochemical reactions across life

• They occupy the widest range of habitats

Nucleoid

The primary genetic material of the cell; the chromosome; circular in most species

Plasmids

• Double-stranded closed circles of DNA

• Exist in multiple copies in the cytoplasm

• Contain one or more genes

  • F factor can integrate into chromosomes

  • R factors carry antibiotic restistance genes

• Replicate independently of the bacterial chromosome

Bacteria are grown in:

• liquid culture medium

• on semisolid agar (petri dish)

Defined minimal medium

• Nutritional components simple

• Organic carbon source (glucose) and inorganic ions

Complete medium, rich medium

Amino acids are added as supplements to minimal medium

Most bacteria are prototrophs

Capable of making (biosynthesis) all amino acids, nucleotides, lipids, sugars, vitamins, etc from inorganic nutrients

Auxotrophs (some bacteria)

Require the addition of a metabolic supplement to their culturing medium in order to grow

Bacteria grow in three defined phases

• Lag phase: initial adaptive phase to favorable growth

• Log phase: rapid cell division, dividing in half to double the cell number exponentially

• Stationary phase: adaption to nutrient depletion or poor growth conditions, expression of stress survival genes

What are the advantages of HGT?

• Allows the recipient organism to explore or exploit a new environment

• Speeds the evolution of the organism

What are some traits that are introduced by HGT?

• Antibiotic resistance genes

• Metabolic determinants

• Virulence determinants

What are the mechanisms for HGT?

• Conjugation: bacterial “sex”

  • Most common

  • Plasmid-mediated; these plasmids are called F+ (fertility)

• Transduction: movement of DNA via a virus

• Transformation: uptake and incorporation of extracellular DNA fragments

Genetic recombination occurs in bacteria

Bacteria are capable of acquiring new genetic material (this is not the entire genome, only part of the chromosome)

rec genes are NECESSARY for RECombination

• Scientists studied defective recombination and found mutant genes; thus, these are necessary for the process to occur

  • Mutations are in genes named recA, recB, recC, recD

  • recA (mutant) strains were completely recombination defective

• These gene products are essential for

  • Recombination during all three of the HGT mechanisms

  • Also important for DNA repair

Conjugation

Definition: temporary fusion of two single-celled organisms for the transfer of genetic material from a donor to a recipient bacterium

• Called bacterial sex

• This process of genetic recombination results in the replacement of one or more genes present in one strain with those genes from a genetically distinct strain

Discovered by the Lederberg and Tatum experiment:

• Prototrophs result from two auxotrophs

Lederberg-Tatum experiment

Lederberg and Tatum (1946) mixed two auxotrophic strains of E. coli, each unable to grow independently on minimal medium because they required different nutrients. When mixed together and plated on minimal medium, some bacteria grew, indicating that genetic material had been exchanged to produce prototrophic recombinants. This provided the first evidence of genetic recombination in bacteria, although the mechanism of conjugation was established through later experiments.

What are the requirements for conjugation?

Cell to cell contact between donor and recipient cells through the F pilus (or sex pilus)

Donor cell with pili (F+), recipient cell (F-)

U-tube experiment showed that physical contact was necessary for

Conjugation

What are the requirements for conjugation?

• The recipient cell lacks the F-factor (cells are called F-).

• The donor cell has to carry the “Fertility factor”, a.k.a. F-factor

  • The F-factor is a self-transmissible plasmid, an episome called the F plasmid.

    • The F-plasmid can exist free in the cell (cells are called F+)

    • The F-plasmid can exist as a segment of DNA integrated into the chromosome (cells are called Hfr).

  • The F plasmid carries genes for

    • Construction of the sex pilus.

    • Replication and transfer of the F plasmid. Only ONE strand of the plasmid is transferred.

Conjugation, F+ x F-

Transfer of the F+ plasmid (and the genes on it)

Very rarely is there transfer of other genetic information (something else is going on)

Both cells are F+ at the end

Hfr donor cells

Hfr (high-frequency recombination)

• Special class of F+ cells that generate more prototrophs when mated (hence the name Hfr)

• They have an integrated F+ in the chromosome

This is a specialied case of F+ mating

The integrated F-plasmid directs

• Formation of the F-pilus

• Transfer of the part of the integrated F plasmid and the adjacent chromosomal DNAs to the recipient strain

  • The recipient gains new alleles of genes that replace homologous portions of its chromosomes

• But, the entire F-plasmid is not transferred

  • The recipient DOESNT become F+

F+ becomes an Hfr by integration

Can Hfr cells mate?

Hfr strains

• Have an F-plasmid integrated into their chromosome

Yes! Similar to F+ cells

• Hfr cells conjugate with F- cells

The F-plasmid directs

• Formation of the F-pilus

• Transfer of the integrated F plasmid and the associated chromosomal DNA to the recipient strain

IMPORTANTLY, the recipient gains chromosomal genes from the donor

Conjugation, Hfr x F-

Lederberg-Tatum Experiment w math

Conjugation can be used to map positions of genes

• Mapping by interrupting conjugation

  • Hfr bacteria with known F+ plasmid genotype are mixed with a strain of F- bacteria of known genotype

  • Conjugation (and gene transfer) commences

  • At set time intervals, a sample of bacteria is taken from the culture and conjugation in that sample is terminated (use a blender), and the Hfr individuals (which have been ‘tagged’ with a trait that allows them to be selectively removed, such as antibiotic sensitivity. This trait is NOT present in the recipient bacteria, so they all survive)

• The longer the time interval, the greater the transfer of the genes into the recipient.

• Because gene transfer starts at the Origin (O) on the integrated plasmid, this permits the determination of the order of genes transferred, genes adjacent to the integration site.

• Remaining F- bacteria are grown in a culture and grown on selective media to determine which phenotypes are newly present. For example, if the investigator wishes to determine the order of genes such as:

  • inability to digest lactose (lac)

  • resistance to sodium azide (azi)

  • sensitivity to T1 bacteriophage (ton)

  • resistance to streptomycin (str)

  • inability to digest galactose (gal)

• Using different Hfr strains, one can map the entire genome.

Using Hfr strains to map genes

• Hfr strains transfer genes at a high rate, starting from the integrated F+ plasmid

• Transfer is only of a portion of the genome, near the site of integration

• Recombination with the chromosome is necessary to capture the new alleles

(in an example, genes a and b must be linked (near each other) because they are both transferred together)

Can the integrated F plasmid be excised from the Hfr chromosome?

• Yes, it can excise exactly as the reverse of the integration (restores F factor)

• Yes, it can excise imprecisely to form an F′ plasmid.

  • The F′ plasmid contains genes from the chromosome on a transmissible plasmid.

  • F′ cells can mate with F- cells.

• When an F′ cell mates with an F- cell the extra genes are carried into the recipient cell and maintained on the plasmid.

  • When there are multiple copies of genes in a cell, the cell is called a merodiploid.

Examples of conjugation in bacteria

• Conjugation can also occur between unrelated species or between kingdoms

  • Example: IncP plasmids can transfer themselves or move other plasmids from E. coli into essentially any gram negative bacterium (heliobacter, pseudomonas, Legionella, etc.)

  • Example: The bacterium Agrobacterium tumefacians can transfer the Ti plasmid (T-DNA) to plant cells where it integrates into the plant genome.

• The transmissible plasmids can carry virulence genes

  • Example: Shigella - carries a >200 kb plasmid that contains genes for cell invasion and adherance.

  • Example: Yersinia species, which cause enterocolitica, pseudotuberculosis and pestis (plague), carry plasmids that encode outer membrane proteins called Yops that may help the bacteria avoid host phagocytosis.

    • Yops are synthesized only under conditions of limiting calcium and high concentration of sodium ions…conditions that may mimic the environment inside a eukaryotic cell.

Transduction

Movement of genetic material mediated by bacteriophage (aka phage)

• Bacteriophage (bacteria eaters) are viruses that infect bacteria

• Genetic material from a previously infected cell can be packed into viral particles, and brought into a new recipient cell

Types of transduction

• Generalized: random DNA fragments are packaged

• Specialized: genetic material adjacent to the integration site of the phage in the chromosome is packaged

The lifecycles of bacteriophage

Lytic phage: only produce additional phage particles and rupture the cell

Temperate phage: two lifecycles with temperate page

• Lysogenic: integrate DNA into host chromosome (they lie low)

• Lytic: enter an active life cycle to produce additional phage particles and rupture the cell to release phage particles

Transduction: generalized transduction occurs with lytic-cycle phage of either type. Specialized transduction occurs only with lysogenic phage that incorrectly excise from the genome

Lytic phage

Only produce additional phage particles and rupture the cell

Temperate phage

Two lifecycles with temperate page

• Lysogenic

• Lytic

Lysogenic (temperate phage)

Integrate DNA into host chromosome (they lie low)

Lytic (temperate phage)

Enter an active life cycle to produce additional phage particles and rupture the cell to release phage particles

Transduction

• Generalized transduction occurs with lytic-cycle phage of either type

• Specialized transduction occurs only with lysogenic phage that incorrectly excise from the genome

Lysogeny occurs when

• The phage DNA integrates into the bacterial chromosome (it is called a prophage)

• Integration requires enzymes that recognize specific places in the bacterial and phage DNAs, cut them, and recombine

  • attB and attP contain short regions of homology

Bacteria containing a prophage are said to be

• lysogenic

• they can grow and divide stably until viral reproduction is induced

  • The phage genome is replicated along with the chromosome and is passed to daughter cells

Generalized transduction

Generalized transduction again

Specialized transduction

DNA adjacent to the integration site is moved to the recipient cell

Why is trasduction important?

• Bacteriophage are agents of microbial evolution and pathogenesis

  • Efficient

  • Phage can carry large pieces of “foreign” DNA

  • Survive harsh relatively harsh conditions

• Virulence determinants are encoded on lysogenic phage and introduced to the bacterium upon infection

  • Ex. toxins expressed in E. coli are carried on the phage genome

Transformation

Taking up of DNA by competent bacteria

• Competence in bacterial cells

  • A small proportion of cells in a population are naturally competent. There are a large number of proteins devoted to this process (DNA uptake, import machinery and recombination).

  • In some cases, e.g. Neisseria gonorrhoeae and Heamophilus influenzae, bacterial species can only take up DNA from the same species.

  • Treatment of cells with salt solutions can make bacteria competent; this is what is done in laboratory settings.

• Extracellular DNA

  • Fragments of DNA (linear); must recombine with chromosome to become stable.

  • Plasmid DNA which is circular and capable of replication is stable; this is what we work with in the laboratory

Transformation: competence in bacterial cells

• A small proportion of cells in a population are naturally competent. There are a large number of proteins devoted to this process (DNA uptake, import machinery and recombination).

• In some cases, e.g. Neisseria gonorrhoeae and Heamophilus influenzae, bacterial species can only take up DNA from the same species.

• Treatment of cells with salt solutions can make bacteria competent; this is what is done in laboratory settings.

Transformation: extracellular DNA

• Fragments of DNA (linear); must recombine with chromosome to become stable.

• Plasmid DNA which is circular and capable of replication is stable; this is what we work with in the laboratory

Note on confusing terminology:

• Transformation is the word used to describe the uptake and incorporation of exogeneous DNA by bacteria, yeast and plants.

• However, the term transformation when used with mammalian cells means the loss of growth control, not the uptake of DNA. That is, transformed cells are cancerous or pre-cancerous.

• The term in mammalian cells for the similar process as bacterial transformation (i.e., taking-up extracellular DNA), is transfection (not to be confused with infection!).

Cells take up DNA fragments and recombine them with the genome

rec proteins are necessary to recombine the new, incoming DNA to replace existing chromosomal information

→ DNA binds to the cell on a receptor

→ DNA strands are separated: one is transferred into the cell and the other is degraded (in the periplasm)

→ the single stranded (ss) DNA is recombined with the chromosomal DNA to make a heteroduplex.

• → double-stranded (ds) but the strands were originally from ds DNA (“hetero”)

→ if there are any differences between the DNA sequences in the heteroduplex

• →if replication happens, the two strands are different, one cell will be transformed

• → DNA repair can fix the mismatch

Plasmids

Contain one or more genes and replicate independently of the bacteial chromosome

Some naturally occuring plasmids:

• F factors confer fertility

• R plasmids confer antibiotic resistance

• Col plasmids encode colicins that can kill neighboring bacteria

Plasmids can also be made through genetic engineering

• These engineered plasmids have an origin of replication, genes to select for the plasmid (ex. in lab)

• Vectors generally carry essential plasmid parts

• Plasmids can be engineered to carry and/or express genes

F factors

confer fertility

R plasmids

confer antibiotic resistance

Col plasmids

encode colicins that can kill neighboring bacteria

Summary

Bacteria use several mechanisms to move genetic material

• Conjugation

• Transformation

• Transduction

These mechanisms play important roles in the evolution of microbial genomes and in the dissemination of various traits such as virulence determinants