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Genetics > Ch. 7 The Genetics of Bacteria and Their Viruses > Flashcards

Ch. 7 The Genetics of Bacteria and Their Viruses Flashcards

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Biology 101 flashcards
1
Q

Eukaryotic genetics

A

Yeast - model system. Single cell fungi.

Genetic mechanisms pretty much same as animal or plant.

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2
Q

Most diverse organisms on Earth

Time for 1 cell division (doubling time)

A

Lab strains in complete media abt 20 min. Everything they need to grow - ideal model organisms.
Strains that live deep under the surface in natural gas fissures abt 10,000 yrs.

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3
Q

Most diverse organisms on Earth

Suitable environments for life

A

Air, water, soil, inside you, lava vents, hot springs, more.

Every environment. Where no other living being can survive.

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4
Q

Methods of growing bacteria in the laboratory.

A

Suspension of bacterial cells (test tube).
Suspension spread on petri plate with agar gel.
Incubate 1-2 days.
Visible colonies (each a clone of the corresponding single cell)
Bacteria colonies: genetic identical clones

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5
Q

Bacteria and viruses as model organisms

A

Small size, short generation time, simple structures.
Clones: allows scientists to rule out individual variation.
Can be grown on distinct culture media-diff nutrients used to grow.
Many phenotypes with underlying hereditary traits.
Distinct culture media can identify nutritional mutants.
Studying bacteria and viruses offered studying genetics on a biochemical level.
Many basic concepts of genetics were first deduced from studies of bacteria and viruses.

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6
Q

Bacteria and viruses in the genomic era

A

Sequencing of over 3500 bacterial genomes have been completed since Haemophilus influenzae in 1995-1st bacteria to be fully sequenced.
Next-generation sequencing has become the predominant mechanism to identify wild populations of bacteria, which is important bc many types do not grow in lab conditions, and are hard to study (microbiome research).
Scientists have started to attempt synthesizing an entire bacteria genome from scratch (synthetic biology)

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7
Q

The genetics of viruses

A

Viruses walk the line between the living and nonliving.
(sometimes classified as living and sometimes non-living).
Can be in dormant state for yrs without eating, reproducing.
Viruses can only reproduce by infecting living host cells (bacteria, plants, animals). (Reproduction- criteria of living beings).
Utilize the machinery of their host cell to express their own genes (do not eat-don’t have metabolism on their own).

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8
Q

Bacteriophages as model systems

A

Viruses that infect and use bacteria as hosts.
In the lab, phages are propagated in bacterial cultures (liquid: broth).
Localized area where the virus have killed the bacteria: plaque (holes).
Categorized into 2 types: virulent (T4) and temperate (lambda).

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9
Q

Bacteriophage T4

A

Whole virus consists of mostly DNA and proteins.
One chromosome: 168,000 bp long with about 150 known genes (abt 150 unknown genes)
T4 is a lytic (virulent) phage

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10
Q

Lytic life cycle

A
  1. Phage is absorbed into bacterial host cell.
  2. Phage DNA is injected; host DNA is degraded.
  3. Phage DNA is replicated; phage protein components are synthesized.
  4. Mature phages are assembled.
  5. Host cell is lysed (dies); phages are released.
    Ex of lytic viruses: influenza, common cold, SARS, rabies
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11
Q

Bacteriophage T4

nucleases

A

Phage-encoded nucleases only destroy host DNA, bc phage DNA has instead of cytosine HMC (cytosine with a CH2OH) with glucose attached. (protects).
Many mutants known: temperature sensitive mutants, size and shape of plaques.
Nucleases- enzyme that degrades DNA - why are they only degrading host DNA and not phage DNA? Phage DNA has a protection mechanism.

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12
Q

Bacteriophage Lambda

A

Double-stranded DNA genome
Genome contains 48,502 base pairs and about 50 genes.
May be lytic or lysogenic (temperate)

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13
Q

Lysogenic/lytic life cycle

A

Lambda phage attaches to bacteria and injects DNA.
Lysogenic pathway - site specific recombination - viral DNA integrated into bacterial DNA. Bacteria divides and DNA propagated and divided.
Lytic pathway - many viral chromosomes, viral assembly, cell lysis.
Prophage can remain in lysogenic stage for very long time, trigger causes it to switch over to lytic pathway.

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14
Q

Site specific recombination

A

Integration of the lambda chromosome into the bacterial chromosome.
Genes carried by lambda can be inserted into bacterial chromosomes.

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15
Q

The bacterial genome

A

One main circular chromosome with a few thousand genes (monoploid, but cells can contain several copies).
Contains one double-stranded DNA molecule with a few million base pairs in length (E.coli 4.6 million).
Variable number of mini-chromosomes: plasmids and episomes (can be integrated in the main chromosomes similar to prophage)
Autonomously replicating circular DNA molecules with 3 to several hundred genes.
90% encodes for proteins in E.coli (in humans, only 1 %)
(Overall genome in bacteria is much more efficiently organized in comparison to Eukaryotes).

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16
Q

Asexual Reproduction in Bacteria

A

Binary fission: resulting daughter cells are clones.
Independent assortment and meiotic crossover are absent in bacteria.
But parasexual processes possible - bacteria can exchange genetic information.
1. Cell replicates its DNA
2. The cytoplasmic membrane elongates, separating DNA molecules.
3. Cross wall forms; membrane invaginates
4. Cross wall forms completely
5. Daughter cells

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17
Q

Growth phases

A 
lag phase (dormant)
Log phase (exponential growth) - doubling mechanism, every cell splitting into 2.
Stationary phase - when nutrients get exhausted, growth slows down.
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18
Q

Nomenclature

A

Genes are named by the phenotype they cause.
-leuA is an auxotroph that requires leucine to grow (auxotrophs need certain nutrients from the environment, they cannot synthesize them).
Wild type gets a superscript+
-leuA+ is the wild type form of the leucine gene
-leu- indicates that the bacteria requires leucine to grow.
Phenotypes are denoted by capital first letter Leu- (genes-small 1st letter).
Genes that provide resistance to a compound a superscript r
-axi^r provides resistance to sodium azide (azi^s sensitive)
-amp^r ampicillin resistant

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19
Q

Echerichia coli (E.coli)

A

The most commonly studies bacterial model system.
Singular, circular chromosome, plasmids.
Lives in the large bowel, can cause disease.
Can divide as quickly as 20 min.

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20
Q

Phenotypes in bacteria

A

Don’t differ by physical appearance of single individual, but usually by colony - color and morphology.
Prototrophs (able to synthesize all metabolites) and auxotrophs (need certain metabolites).
Nutritional mutants for energy sources (wild type E.coli is phenotype Lac+ and genotype lac+).
Antibiotic resistance mutants.

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21
Q

Selection screen to identify phenotypes

A

Commonly used to identify bacteria with recombinant plasmids.
The plasmid contains a gene that will allow the bacteria to survive only if it keeps this plasmid (ex: antibiotic resistance).
In the lab: plasmid contains a gene for ampicillin resistance; bacteria are grown on a plate with LB/Amp medium; only bacteria which picked up the plasmid are able to grow on this plate.

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22
Q

Selection screens

A
  1. Invert master plate; pressing against velvet surface leave an imprint of colonies.
  2. Invert 2nd plate (replica plate); pressing against velvet surface picks up colony imprint.
  3. Incubate plate.
  4. Only penicillin-resistant colonies grow. Compare with position of colonies on original plate.
    All bacteria grow on the master plate; however only mutants with a selective advantage will survive on the selection plate.
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23
Q

Genetic recombination without sexual reproduction

A

Provides basis for development of chromosome mapping methodology in bacteria.
Genetic information is transferred.
Results in altered genotype (and phenotype).

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24
Q

Gene Transfer

A

Vertical and horizontal

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25
Q

Vertical gene transfer

A

Transfer of genetic information b/t members of same species

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26
Q

Horizontal gene transfer

A

Transfer of genetic information between related but distinct species.
Plays a significant role in evolution of bacteria.

27
Q

Unidirectional gene transfer leads to recombination

A

In eukaryotes, genes are exchanged reciprocally during meiosis: recombination happens b/t two complete chromosomes. (leads to genetic info transfer, genetic recombinant offspring).
In bacteria, gene transfer is unidirectional: Recombination occurs between a fragment of one chromosome (from a donor cell) and a complete chromosome (in a recipient cell).
Crossover must occur in pairs and must insert a piece of the donor chromosome into the recipient chromosome.

28
Q

Two crossovers

A
  1. Gene transfer in bacteria produces a partially diploid recipient cell containing a fragment of the donor cell’s chromosome.
  2. Two crossovers insert a segment of the donor cell’s chromosome into the intact circular chromosome of the recipient cell. The replaced recipient fragment is degraded.
    Integrity of the circular chromosome is maintained
29
Q

One crossover

A
  1. Gene transfer produces a partially diploid bacterium..
  2. A single crossover between the linear donor fragment and the intact circular chromosome of the recipient produces an unstable linear chromosome.
    Integrity of the circular chromosome is destroyed, a linear chromosome is produces which is unstable (cannot replicate and will degrade).
30
Q

Mechanisms of Gene transfer in bacteria

Bacteria exchange genetic material through three different parasexual processes:

A

Transformation
Conjugation
Transduction

31
Q

Transformation

A

uptake of free DNA

32
Q

Conjugation

A

direct transfer of DNA from one bacterium to another

33
Q

Transduction

A

transfer of bacterial DNA by a bacteriophage

34
Q

Which process is responsible for recombination?

A

DNase (deoxyribonuclease): enzyme which degrades DNA; if adding DNase interrupts the gene transfer, transformation is involved bc the free DNA willl be degraded; in conjugation and transduction the DNA is protected within the bacteria or viruses

35
Q

The U-Tube Experiment

Test for cell contact

A

Bacteria of different genotypes are placed in the tube, separated by a glass filter to prevent direct contact. If recombination occurs, it cannot be due to conjugation but must be due to transformation or transduction.

36
Q

Transformation, Conjugation, Transduction

A

Species dependent: the 3 parasexual processes do not necessarily occur in all bacteria species.
Transduction (being infected by a virus) is potentially the only process occurring in all species.
Certain genes and metabolic machinery are required for transformation and conjugation:
For ex, natural populations of E.coli do not posses the genes required for transformation (there are lab methods to make E.coli ‘competent’, and there are lab strains of competent E.coli.

37
Q

Transformation discovery

A

1928: Frederick Griffith discovers transformation in Streptococcus pneumonia.
Colonies with capsule’s - virulent. Capsule prevents immune system from recognizing the bacteria and attacking them.
Colonies without capsules - avirulent. Don’t cause pneumonia.
Bacteria can transfer genetic info from one individual to another.

38
Q

Griffith’s Experiment 1928: the ability to form capsules is transferrable

A

Living type S (capsule) injected = dead mouse. Living type S recovered.
Heat-killed type S injected = live mouse.
Living type R (no capsule, avirulent) injected = live mouse.
Heat killed type S and living type R injected = dead mouse. Living type S bacteria recovered.
Showed the ability to form capsules must be genetic info, can be transferred from one type of bacteria to another.
Demonstrated that the new ability is passed onto daughter cells. Therefore it must be due to genetic change.
That set the stage for the demonstration that indeed DNA is the carrier of genetic information.

39
Q

Experiments by Sia and Dawson 1931

A

Showed that the phenomena, now called transformation, was not mediated by a living host.

40
Q

Mechanism of Transformation

Competency

A

Competency- not all bacteria are capable to be transformed.
Only competent bacteria can be transformed.
Competent bc they have specific protein channels in cell wall or we can make them competent in lab by adding ice cold Ca.

41
Q

Competent bacterium

A

Let DNA through its cell wall and membrane.

  1. Extracellular DNA binds to the competent cell at a receptor site.
  2. DNA enters the cell, and the strands separate.
  3. One strand of transforming DNA is degraded; the other strand pairs homologously with the host cell DNA.
  4. The transforming DNA recombines with the host chromosome, replacing its homologous region, forming a heteroduplex.
  5. After one round of cell division, a transformed and a nontransformed cell are produced.
42
Q

Outcome of Transformation

A

2 steps of transformation
1.Entry of foreign DNA into recipient cell.
2. Recombination between foreign DNA and homologous region in recipient chromosome.
First step alone results in additional foreign DNA to cytoplasm but not chromosome.
Completion of both steps required for genetic recombination.

43
Q

Transformation efficiency

A

LB plates: nonselective; transformed and untransformed colonies can grow.
LB/Amp plates: selective; ampicillin resistance is used as indicator of successful transformation. Only colonies which integrated the plasmid can grow on LB/Amp plates.

44
Q

Transformation

Super-bugs

A

Can spread resistance genes in bacteria populations and can lead to the emergence of “super-bugs” such as MRSA (Methicillin-resistant staphylococcus aureus).
Today one of the most common used techniques in bioengineering.

45
Q

Conjugation discovery

A

1946: Lederberg and Tatum discover gene transfer by conjugation in E.coli.
Some bacteria can build appendages F pilus-use them to get into contact with other cells.
2 bacteria get into physical contact with each other through a protein channel.

46
Q

The F factor (fertility) in E.coli

A

F- cell doesnt have ability to form F pilus. (recipient)
Only donor cells have F pili: appendages to make contact with recipient cell (lack F pili and F factor F-).
The synthesis of the F pili is controlled by the F factor.
The F factor can be autonomous (F+) or integrated (Hfr cell).

47
Q

Conjugation with Pilus

A
  1. Conjugation occurs between F+ and F- cell.
  2. One strand of the F factor is nicked by an endonuclease and moves across the conjugation tube.
  3. The DNA complement is synthesized on both single strands.
  4. Movement across conjugation tube is completed; DNA synthesis is completed.
  5. Ligase closes circles; conjugants separate. Now have 2 F+ cells
    The recipient F- cell is converted to F+.
    During conjugation, the DNA of the F factor is replicated, with one new copy entering the recipient cell, converting it to F+.
48
Q

Formation of Hfr cell

A

Hfr= high frequency recombination
Several sites suitable for integration.
Then integrated the F factor mediates transfer of the chromosome during Hfr-F-mating.
Most often mating and transfer is interrupted, only rarely an entire chromosome transfers.
(F+ can be converted to Hfr).

49
Q

F plasmid can integrate into multiple locations

A

Resulting in different Hfr strains

50
Q

Mechanism of transfer

A

Same mechanism in both Hfr cells and F+.
Transfer initiates at the oirT site (origin of transfer) through DNA replication.
oriV and oriS initiate DNA replication during cell division (but not during transfer).
Rolling circle replication (DNA replication in bacteria) - one copy of the chromosome is synthesized in the donor cell and the transferred strand of donor DNA is replicated in the recipient cell.

51
Q

Using conjugation to map E.coli genes

A

Conjugation between Hfr and F- cells has been used to map genes on the E.coli chromosome.
Hfr and F-cells with certain mutant genes are mated.
The mating is interrupted in certain time intervals.
Depending on these time intervals and the position of genes on the chromosome, the genes have been transferred yet or not.

52
Q

Interrupted mating experiments

A

Genes are arranged linear on the circular chromosome.
Transfer starts at oriT.
Mating is interrupted in certain time intervals.
Cells get plated on selective media.
Genes closer to oriT get transferred earlier.
The more time passes the more of the chromosome (and the more genes) are transferred.

53
Q

Time mapping

A

you can come up with time map after interrupting at certain times.

54
Q

Linkage map of E.coli

A

Transferring the complete chromosome takes 100 minutes.
Map distance of 1 minute corresponds to the length of segment transferred in 1 minute.
Different Hfr strains: oriT is not always on the same site.
Orientation can be clockwise or counterclockwise.

55
Q

Plasmids

A

Double-stranded closed circles of DNA.
Exist in multiple copies in cytoplasm.
Contain 1 or more genes.
Replicate independently of the bacterial chromosome.
Most plasmids are not required for the survival of the host cell (no essential genes).

56
Q

3 major types of plasmids in E.coli

A 
F factor (Fertility factor)
R plasmids (Resistance Plasmids)
Col plasmids (colizines; proteins toxic to bacterial strains that do not harbor same plasmid; not transmissible to other cells)
57
Q

R plasmids

A

2 components:
RTF - resistance transfer factor (can transfer resistance info onto another cell - bad for antibiotic resistant bacteria).
r-determinants TC, tetracycline; Kan, Kanamycin; Sm, streptomycin; Su, sulfonamide; Amp, ampicillin; and Hg, mercury. Caries info for antibiotic resistance - diff types, the more r-determinants the harder the bacteria is to kill.

58
Q

Formation of an F’ Factor

A
  1. The F factor loops out of the chromosome with the thr and leu genes in the loop.
  2. A crossover excises the F factor carrying the thr and leu genes, producing an F’ thr leu.
    By integrating an F’ factor into the chromosome a cell becomes Hfr. This process can sometimes work in the other direction: A Hfr cell produces a F plasmid
59
Q

Transduction

A 
A bacteriophage (virus) transfers DNA from a donor cell to a recipient cell.
Possible in all kinds of diff bacteria bc of virus infection
60
Q

Generalized transduction

A

A random fragment of bacterial DNA is packaged in the phage head in place of the phage DNA.

61
Q

Specialized transduction

A

Recombination between the phage chromosome and the host chromosome produces a phage chromosome containing a piece of bacterial DNA.

62
Q

Normal Excision of lambda Prophage

A

1.The lambda prophage loops out and attBP pairs with attPB.
2. Site-specific recombination occurs between attBP and attPB, excising the phage lambda chromosome.
Prophage cut itself out and is a lambda chromosome. Can be transformed from 1 cell to another.

63
Q

Anomalous Excision of lambda prophage

A
  1. The lambda prophage loops out anomalously with attBP and attPB not paired.
  2. Recombination excises a lambda chromosome carrying E.coli gal genes, and lambda genes are left in the E.coli chromosome.
    Anomalous excision of the lambda prophage produces a lambda dgal transducing chromosome.
    (Cut out piece of the bacteria chromosome)
64
Q

Generalized transduction steps

A

Process of transduction where bacterial recombination is mediated by bacteriophage.
Random DNA fragment gets accidentally packaged into a virus body and transferred to another bacterium.
1. Phage infection
2. Destruction of host DNA and replication synthesis of phage DNA occur.
3. Phage protein components are assembled.
4. Mature phages are assembled and released (phage carrying a piece of bacteria DNA in head)
5. Subsequent infection of another cell with defective phage occurs; bacterial DNA is injected by phage.
6. Bacterial DNA is integrated into recipient chromosome.

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