Test 3 Biol 518

Bacterial Natural Transformation

A form of genetic exchange in which free DNA is taken up by a bacterial cell, and stably maintained.

What does maintenance of bacterial natural transformation require?

• homologous recombination into the chromosome

• mainenance as an extra-chromosomal element (ie- a plasmid)

competent

cells capable of DNA uptake

naturally competent

naturally capable of competence (not all are)

Discovery of competence

DNA is hereditary material. Discovered by Griffith, which later led to the discovery that the "transforming principle" was in fact DNA.

(T/F) The implementation of natural competence has evolved to be the same as the gram-positive and gram-negative species.

False, it has evolved to be different.

Steps of natural transformation:

• DNA binding to outer surface (G- OM, G+ CW)

• DNA uptake: movement across OM/CW

• Degrade one strand

• ssDNA across IM into cell cytoplasm. Complement strand synthesized/plasmid. Integrate into chromosome. Degraded.

Difference between G+/- transformation:

• G+: Specificity due to competence only in presence of related bacteria (Quorum sensing)

• G-: Specificity due to sequence specificity of DNA binding (DNA- Uptake Sequence, for example)

(T/F) anything that is brought into the cell is coded by RecA.

True

What type of pili does type IV secretion have?

Type II pili

What type of secretion assembles Type IV pili?

Type II

(T/F) Pili and secretion systems are the same.

False, they are not the same.

Steps in Gram-positive transformation:

1.) DNA binding to the cell surface. dsDNA can bind at the cell surface. It is not sequence specific.

2.) Entry of the DNA into the cell wall. DNA passes through or near Type IV pilus.

3.) Passage of the DNA through the cell membrane. As DNA passes through the cell membrane, strands are separated and a competence-specific nuclease degrades one strand. ssDNA enters the cytoplasm and is bound by SSB/ other protein.

(T/F) Only double stranded DNA can bind in gram-positive transformation.

True

Steps of Gram-Negative Transformation:

1.) Competence. Some are constituitevly competent. Does not involve protein pheromones.

2.) DNA binding. Binds ssDNA. DNA sequence specific (DUS)

3.) DNA entry. Type IV pilus (most common, and similar to G+ system, but with OM channel). Type II secretion system. Transformasomes (membrane associated vesicles that take up dsDNA).

4.) Establishment of DNA in recipient. Homologous recombination.

How did scientists learn about the mechanisms involved in competence?

They used DNA uptake assays. They would radio label DNA, add DNA to cell, and as it entered, they would track the radio label as it entered the cell.

Steps of DNA uptake assay:

1.) Mix Arg+ DNA and Arg- recipient cells.

2.) Treat mixture with DNase at various times.

3.) Extract DNA and mix with Arg- recipients.

4.) Select Arg+ transformants.

(T/F) The rate of co-transfer determines how close in proximity two mutations are.

True

(T/F) In co-transfer, two mutations that are close in proximity will travel together (when transferred to a new bacterial strain).

True

What can co-transformation be used for?

It can be used to identify linked markers.

• Two markers may be cotransduced if they are near each other in the chromosome.

• The proximity to each other will correlate with the frequency that the markers are cotransduced.

• Before next-gen sequencing, this approach was useful to identify where mutations were in the genome (to 'map' mutations)

Congression

Refers to when two unlinked genetic markers enter the cell at the same time through different sites (they appear to be co-transformed and linked but really are not).

(T/F) Congression occurs because there can be more than one site of entry for the DNA.

True

Artificial Competence

• Cells are passively made permeable to DNA, by artificially creating favorable conditions that would otherwise not normally occur.

• Chemical transformation, electroporation, protoplasm transformation

• Can be used to take up: plasmid DNA, phage DNA, chromosomal or linear DNA

Natural Competence

• Process by which bacteria naturally take up DNA

• Chromosomal DNA is most efficiently taken up by natural competence

• Why? natural competence requires breakage and degradation of one of the strands. Plasmids and phage must be cyclized and double stranded after entering cell (can happen but it is low efficiency!)

• Gram+ induce natural competence in response to neighboring population density

• "Quorum sensing," which activates behavioral changes when the population reaches a sufficient density or "quorum."

Quorum Sensing

• The ability of bacteria to sense the presence of other bacteria via secreted chemical signals.

• Controls many things like: conjugation (recipient cells emit signal that indicates a quorum), light production, and competence (early studied system)

• Each system uses different mechanisms but all have the same outcome: activates transcription of certain genes at specific population density

Regulation of Competence Development in Bacillus Subtilis:

• "Com" = competence defective

• ComX is the Pheromone

• When the ComX pheromone binds to ComP at the cell surface, initiates cell signaling that results in expression of competence genes

• ComP is a sensor histidine kinase (= can phosphorylation other proteins to activate them)

• ComP phosphorylation ComA

• ComA is a response regulator that activates gene transcription

• ComA also activates ComK, another transcriptional activator

Regulon

Any set of opera songs and genes regulated by a regulator

How does peptide signal respond to population of neighboring cells?

Secreted continuously, such that peptide concentration in the environment corresponds with density of cell population. Signal is processed to active form during export, thus preventing cells from responding to their own peptide signal

Why are bacteria naturally competent?

It allows for horizontal transfer allows for antibiotic resistance, etc.

3 Hypotheses of why bacteria are naturally competent: Nutrition

DNA could be a carbon source!- extra source of food in tough times

3 Hypotheses of why bacteria are naturally competent: DNA repair

• Useful to fix damaged DNA with neighbor's DNA.

• Process: UV irradiation induces damage to DNA. Some bacteria die, releasing their DNA. This DNA is taken up by other bacteria and replaces their damaged DNA by recombination.

Which of the 3 hypotheses is correct in regards to why bacteria are naturally competent?

Jury's out! There is evidence for and against all three possibilities, so could be they all play a small role or that it is different for different circumstances.

Which of the following about transformation is true?

a.) Ultimately, both strands of a piece of DNA are brought into the cell

b.) All known naturally competent species are descended from a single naturally competent ancestor

c.) Natural competence can be induced by quorum sensing in both gram negative and gram positive bacteria

d.) Once in the cell, transformed DNA may be degraded

d.) Once in the cell, transformed DNA may be degraded

Outline briefly the reasoning used to identify competence for the first time (in 1928- Griffith's experiment)

a.) Heat-killed type S and live type R resulted in live type S infection due to DNA transfer

b.) Heat-killed type R and live type S resulted in live type R infection due to DNA transfer

a.) Heat-killed type S and live type R resulted in live type S infection due to DNA transfer

If you had a plasmid with NO ANTIBIOTIC RESISTANCE MARKER can you still do a transformation?

Yes

Do you think a smaller plasmid will transform better than a bigger plasmid?

Could you transform two plasmids at the same time? (assuming they are compatible)

(T/F) DNA is very tightly packaged in phage head.

True

(T/F) No phages are dsDNA viruses.

False, nearly all phage are dsDNA viruses.

Bacteriophage three families have:

• Complex contractile tail (T4)

• Short noncontractile tail (P22)

• Long noncontractile tail (P2)

Why are phages the most important tool for early molecular genetics?

• Haploid (easy to make mutant crosses)

• Easy to grow a lot of them

• Easy to store

• Helped us determine basic molecular genetic principles of all life

Host range

Number of bacterial species a phage can infect (usually limited)

Permissive host

Bacterial strain that allows infection of a particular phage

Conditional-lethal mutations

Phage mutations that allow growth in one condition but not another e.g. low but not high temperature; very useful experimental tool

Permissive conditions

Allow phage to multiply

Nonpermissive conditions

Do not allow phage to multiply

MOI

Multiplicity of infection (#of phage infecting each bacterial cell)

Phage λ

The model phage

Lytic

Virulent infection- ends with cell lysis

Lysogenic

Quiescent prophage coexisting with bacterial host

Steps of Bacterial Lytic Infection:

1.) Attachment

2.) Entry- usually only DNA

3.) Production of phage protein and DNA

4.) Phage assembly

5.) Phage release

Lytic Infection: Attachment

• Phage attaches to receptor on surface of bacterial cell

• Receptors are proteins or carbohydrates- serve a function for bacterium

Lytic Infection: Entry

• Usually only DNA enters cell

• Some phage package one or a few proteins that also enter cell

• Tailed phage- DNA directly enters cell through tail (syringe-like)

Lytic Infection: Produce Phage Proteins and DNA

• Shut down bacterial processes

• May cut bacterial DNA into fragments

Lytic Infection: Protein Synthesis

• First, bacterial RNAP transcribes phage genes

• Later bacterial RNAP may be modified, or phage-specific RNAP (or sigma) may be made

• Gene expression carefully timed so genes expressed as needed

Timing of Phage Gene Expression:

• Phage genes are often expressed in a regulatory cascade:

• A product of 1st set of genes turns on expression of 2nd set

• A product of the 2nd set, turns on expression of 3rd set

• Gene products sometimes also block earlier or later gene expression

Phage λ Transcription Antitermination

• without N, RNAP terminates at tR1 and tL1

• Once N accumulates: N binds to the nutR and nutL sites in the mRNA. N then interacts with RNAP. Then, N modifies RNAP so it won't recognize the transcription terminators

• The early genes will be transcribed

• E. Coli NusA, B, E, G also required

• Similar to Rho transcription termination, but OPPOSITE effect.

Anytime you see a P it refers to:

A promoter

Anytime you see a T it refers to:

A terminator

Lytic Infection: DNA Synthesis

• Inhibit bacterial DNA replication, often by degrading bacterial DNA

• Promote phage DNA replication, sometimes by making a phage-specific DNA polymerase

λ Replication

• DNA linear inside phage head

• cos sites= complementary

• ssDNA overhangs- circularized DNA in cell

• First replication is theta replication

• Then rolling circle replication

• Generates long "concatemers"

Concatemers

• multiple λ genomes long

• this DNA is packaged, one genome length per phage

Lytic Infection: Phage Assembly

• Phage heads and tails assemble separately

• Additional proteins may be required for assembly, but largely self assembly

• DNA packaged into phage heads

• Tails are attached

Phage Release

- Some phages leak out gradually, leaving the host alive, but most phages nuke them leaving behind nothing that respires

Lytic Infection: Phage Release

• Usually two phage proteins involved:

• Lysozyme/ Edolysin- degrades peptidoglycan, causes lysis

• Holin creates a pore in the inner membrane so lysozyme can get to the peptidoglycan

(T/F) Phages replicate like plasmids.

True

λ Holin: Antiholin

S107 is an antiholin that prevents the S105 holin from forming a pore. At time of lysis the membrane properties change allowing S107 to release S105 and both form pores

Lysogenic Infection

• Phage DNA stably maintained in bacteria

• For most phage, DNA integrates into bacterial genome= prophage (at left)

• For other phage, DNA maintained in a plasmid-like state

• Phage must turn off lytic genes and integrate DNA (if relevant)

λ DNA Integration

• Site specific recombination: Catalyzed by specific proteins at specific sites, very little DNA homology

• λ contains attP site, POP' (240 bp, P for phage)

• E. Coli genome contains attB site, BOB' (25 bp, B for bacteria

• Only homology is 15 bp "O" of sites

• λ integrase (Int) protein promotes the recombination

• E. Coli IHF protein (integration host factor) assists

• Excision also requires λ Excise (Xis) protein

Induction of λ Lysogens:

• Induced by DNA damage

• ssDNA from damage binds REcA; RecA* (RecA nucleoprotein filament)

• Cleaved cl can't repress pR and pL promoters

• Lytic genes are expressed

• Excision is reversal of integration (Requires integrase (Int) and IHF)

• Excision also requires λ Excise (Xis) protein

Phage λ Lytic/ Lysogenic Decision

• λ can undergo lytic or lysogenic infection

• Environment in the bacterial cell determines whether cll is stable

• If stable, cll activates int and cl gene expression; lysogenic infection

• In unstable, pR and pL active, lytic infection

Transduction

Phage-mediated transfer of bacterial DNA sequences

Generalized Transduction

• The particles carrying bacterial DNA form during a lytic infection

• Errors in DNA packaging during a lytic infection

• Remember, phage often cuts bacterial DNA into fragments

• Fragments of bacterial DNA are packaged instead of phage DNA

Specialized Transduction

• The particles carrying bacterial DNA form after a lysogenic infection

• Result from mistake in excision of lysogen

• Only contain regions of bacterial DNA next to prophage integration site

• Contain both phage and bacterial DNA

• A population of identical transducing particles can be made

Mechanism of Generalized Transducton:

• Lytic infection

• Injects DNA

• Cleaves host DNA

• Make phage heads and tails

• Rare mistake: Bacterial DNA packaged instead of phage DNA

• Transducing particle then injects DNA into a new bacterium

• Bacterial DNA is integrated by homologous recombination

(T/F) Many bacteria have "Adaptive immunity" systems that prevent integration of foreign DNA as protection- e.g. CRISPR (cluster of regularly interspaces short palindromic repeats.

True

(T/F) CRISPR systems are restriction modification systems that recognize and cleave foreign DNA based on sequence.

True

Adaptive Phase

PAM sequence is integrated into spacer site in genome

PAM

Protospacer adaptive motif

Cas

CRISPR associated antivirus response

Immunity Phase

• Spacers are expressed as a long mRNA, which are chopped up to make "guide" RNAs.

• Guide RNA pairs with incoming phage DNA and directs CRISPR/Cas system to cleave phage DNA

operator sequence

where a repressor can bind and block RNA polymerase action

(T/F) We don't always understand what leads to lytic vs. lysogenic situations.

True

(T/F) If C2 is very active, it will lead to the production of C1, which will lead to lysogeny and the representation of the lytic cycle.

True

In a scientific paper, the first author listed is:

The person who did all the work

Where to begin when reading a scientific article:

Read the abstract

What is not a way of inducing artificial competence?

a.) Chemical transformation

b.) Electroporation

c.) Using bacteriophage

d.) protoplast transformation

c.) using bacteriophage

Bacteriophage Lambda regulates the expression of its lytic genes in a cascade resulting in very early, early, and late categories, by expressing proteins during the very early and early stages of the infection accumulate and result in the expression of the early and late genes, respectively, by functioning as:

- Transcriptional anti-terminators that enable early and late gene expression, respectively

In lambda phaeg, the decision to become lytic or lysogenic is due to competitionbetween which two proteins?

a.) CI and N protein

b.) CI and CII

c.) Cro and N protein

d.) Cro and CI protein

d.) Cro and CI protein

Barbara McClintock

• Won the Nobel Prize for discovering mobile genetic elements (first woman to win it unshared)

• American geneticist (born in 1902)

• She studied corn and their hereditability of colors through transposons

Transposons:

• Considered a parasite (Similar to plasmids and phages) and are by themselves detrimental although they can also encode helpful elements such as antibiotic resistance genes

• Frequency of transposon is RARE (1 in 10^3 to 10^8 cell divisions- the higher end is similar to rate of mutation through replication alone)

• It is thought that over half of our DNA is made up of transposons and that they have made a big contribution to evolution

how does transposition occur?

Transposon leave donors and move into target. Sometimes it is copied on its way over. This is called a transposition event

Minimum requirements of transposons:

• Inverted repeated ends

• Genes encoding transposons inbetween those^

Types of Bacterial Transposons:

• Insertion Sequence

• Composite Transposon

• Noncomposite Transposon

Insertion Sequence

Transposon with just the minimal requirements

Composite Transposon

Two IS elements of the same type form a larger transposon

Noncomposite Transposon

The enzymes that promote transposition are between two IR elements with no insertion sequence

(T/F) IS elements can transpose DNA between them

True

Assembly of Plasmids by Insertion Sequence and Composite Transposons:

• ISs often surround antibiotic resistance genes

• R-factor: small plasmid that confers resistance to multiple antibiotics

• Transposons may have played a major role in producing naturally occuring multi-resistance plasmids

Nonhomologous recombination

Recombination that does NOT depend on sequence homology (e.g. different from homologous recombination in that it does not involve extensive base pairing)