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Genetic Variation (bacteria)

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What is the nature of sponatenous mutation?
When transcription makes an error – sequence is not exactually how it should be.
= SPONTATENOUS MUTATION
= Intrinsic malfunction – happens from and because of the DNA/organism itself
What is the nature of induced mutation?
When the DNA is damaged after the fact by chemical or physical agents.
= INDUCED MUTATION
= Extrinsic factors affecting the DNA (like different environmental substances)
What is the main difference between bacteria and most organisms in reference to mutations?
When most organisms encounter a mutation in their cell(s) it does not necessarily mean that it will be passed on to the organisms “children”. The type of reproduction used by the organism is usually predictive of this – humans: unless mutation happens in sperm or egg cell than it will not be inherited; plants must undergo mutation in their seed DNA or it will not be inherited.
BACTERIA are almost all unicellular and divide by binary fission (nonsexual reproduction)…so when their DNA is changed it is ALWAYS inherited by the next generation. Henceforth, it is impossible to ever have a pure culture of Bacteria because spontaneous mutation always occurs and is always passed on….they are constantly changing. The bacterial gene is haploid, so there is no second copy of the gene to mask the effect of mutations.
What is a mutant?
Mutant is the name for the organism of the next generation that has obtained altered DNA (different from parent) due to a mutation.
What does progeny mean?
Progeny is the descendent of a parental cell⬦. It will become parent of its descendents which will be its progeny.
What does the term wildtype refer to?
Wildtype specifies the original genotype (genetic makeup of organism) and phenotype (what an organism is physically like as a result of its genotype) of an organism when FIRST isolated in nature (sampled).
What is the difference between constitutive and induced production of a protein?
When a bacteria produces proteins or other gene products in a continuous way, they are said to be CONSTITUTIVE. On the other hand, gene products produced in response to a stimulus are said to be INDUCIBLE.
What is an operon?
When related genes of a bacteria control similar/related functions, they are sometimes organized as operons (groups of genes all controlled by the same regulatory factors); this allows their functions to be coordinated and more effective.
What are the genes that control and repress lac metabolism?
Three genes control lactose metabolism: lacZ, LacY and LacA. They share one promoter and are transcribed as a single message.
The gene encoding the repressor protein to these genes is called the lacl gene and is beside the lac operon. It is transcribed constituitively, and has its own promoter.
What is the regular state of the lac operon and what can affect it?
The basal (normal) state of the lac operon is a repressed one, as the represser proteins produced by the lacl gene bind to the lac operon promoter and mostly block transcription.
When lactose is present, transcription is INDUCED in the lac operon. Lactose binds to the repressor proteins and they are no longer able to bind to the promoter.
What are two mutations that affect expression of the lac operon?
1)a mutation in lacl results in the proteins that repress lac operon to be non-functional…they don’t bind and the lac operon is expressed constituively.

2)A mutation in the operator sequence (called Oc) of lac operon can disallow the repressor proteins (functional) to bind…this also results in constitutive expression.
What is phenotype completely dependent on?
Phenotype is completely dependent on the collection of proteins existing and being expressed in an organism. Each protein has a distinct function, which is due to the unique amino acid sequences in its structure.
How does mutation affect the phenotype/genotype of a bacteria?
Mutation has the ability to change or add characteristics of a cell or organism through the alteration of its genes which can affect the kinds and types of protein that are expressed.
It always changes the genotype (one base pair is considered a mutation) but not necessarily the phenotype.
What determines whether the phenotype will be changed?
The determining factor is how the amino acid sequence is changed and where.
How does mutation affect the non-coding regions differently?
Mutations in the non-coding region can also affect the phenotype of a protein, but in a different way. Because this could happen in non-coding regions such as the promoter, the amount or degree that the protein is expressed could be affected. If a mutation increases the strength of a promoter sequence, RNA polymerase will bind tighter and henceforth produce more RNA/protein; if it decreases it than less will be produced
What is usually the effect of mutation on bacteria and why?
Bacteria are finely tuned to their environment because they are so succeptable to mutation; over millions of years natural selection (the survival and reproduction of organisms who’s characteristics make them better suited to the environment and more likely to survive and pass on their characteristics) contributed to the best form possible of bacteria in each environment. So any mutations that occur now are usually not beneficial and are often harmful.
What does it mean when a gene of an organism is referred to specifically (ex. LacZ)?
When the genotype of a strain of organisms is expressed specifically this is telling us that there is a mutation in this particular gene of the organism and that it is the only one with a mutation (rest unchanged). LacZ + = wildtype while LacZ - = mutant.
What does it mean when the phenotype of an organism is specifically expressed (ex. Lac +)?
When the phenotype is expressed specifically as Lac + or Lac -, this is telling us that the function of this characteristic/ability is either present or not present. Usually (+) = wildtype and (-) = mutant.
What does semi-conservative mean?
the daughter chromosome of each template strand contains one preexisting strand from the parent and one newly made strand.
What happens when a base substitution mutation takes place (in the first and subsequent generation)?
When a mistake is made, the first generation maintains the incorrect pairing (ex. A with G) and it is held together by the H-bonds of the surrounding nucleotide pairs. In the next generation the mutation is STABILIZED by a correct matching of the wrong nucleotide, which results in the next generations inheriting the mutation.
What happens when the template strand slips during replication?
When slippage occurs, a loop of the DNA strand forms and is held in place by H-bonds that are surrounding it; it occurs when there is a REPEATING SEQUENCE usually. When the template strand slips, an insertion occurs to match the ‘extra’ nucleotide.
What happens when the newly synthesized strand of DNA slips?
Another type of spontaneous mutation is the loss or gain of nucleotides resulting from DNA strand slippage. When the newly synthesized strand slips, a deletion occurs, so the other strand deletes a nucleotide to match.
What is a frameshift mutation?
A change in the codon reading frame.
What is a point mutation and what are the two types?
A POINT MUTATION involves a change in a single base pair. Either a TRANSITION MUTATION (a pyrimidine is replaced by the other type of pyrimidine or a purine by the other) or a TRANSVERSION MUTATION (a purine is replaced by a pyrimidine or vice versa) can occur.
What is a polar mutation and which types can cause it?
A POLAR MUTATION is any mutation that affects the transcription/translation of a gene from downstream of the mutant site. Ex. NONSENSE MUTATIONS (cause protein to end prematurely during translation) and FRAMESHIFT MUTATIONS (the insertion or deletion of nucleotides causes a shift of reading frame so that the sequence is read incorrectly).
What is the process for a bacteria aquiring new DNA into its own genetic information?
For the DNA to get into the recipient cell, it must bind to the cell wall, which contains a specific RECEPTOR PROTEIN that transports it inside. One major thing that happens during this transport is the destruction of one of the strands of DNA by a DNase (a specific exonuclease that cleaves nucleotides from the free end of a strand); this results in only a single strand of DNA entering the cell. There is the possibility that this strand will also be degraded inside; however the strand can also get integrated into the recipient’s DNA by homologous recombination (exchange of DNA fragments between two chromosomes).
What is the competent state and how does it differ among bacteria?
Bacteria differ in the state in which they can accept different DNA. Some must be in a COMPETENT STATE (a special physical state), which they reach through some kind of nutrient limitation (they reach this state in late log or stationary phase). Others are always in this state and therefore always capable of gene acquisition. In order to intake or transport “naked DNA” into the cell, a specific set of proteins are needed; these are called COMPETENCE (Com) proteins that are made by the cell when it is in competent state
What is an uptake sequence and why is it required by some bacteria?
Bacteria differ in their ability and tendency to receive genetic information. Some will incorporate any DNA, bacterial or not. Others are very specific and only bind and transport DNA of bacteria that is closely related to them; they recognize their “relatives” by the assistance of a special nucleotide sequence contained in the fragment of DNA, called an UPTAKE SEQUENCE. It is found in multiple random locations throughout chromosomal DNA; the fragment will only be bound and transported if it contains this sequence.
What are the two things that can happen to DNA that is transported into a bacterial cell?
1) It can be degraded by exo/endonucleases (degrading enzymes) that exist specifically to degrade single stranded DNA. This is what almost always happens.

2) It can become coated with a special protein – RecA – that protects it from degradation. This leads to the potential for the donor DNA to be taken (integrated) into the recipient chromosome; however, often the bacteria requires a high degree of homology (genetic similarity). When the recipient and donor share a small stretch of nucleotide information (a few hundred) there is then the potential for homologous recombination (exchange of DNA fragments between two chromosomes at a site of identical sequence of nucleotides).
What is recombination?
This refers to the breaking and joining of DNA strands; the process is instigated through the help of RecA, which promotes invasion of the double stranded (recipient) DNA by the single stranded (donor) DNA.
What are the two other important proteins (enzymes) involved in recombination and what do they do?
Nickase: this is a specific endonuclease that acts as scissors to cut out the recipient strand by breaking the DNA sequence one bond at a time. Break is made at a sequence called the X (chi) site.

Ligase: this is the complementary enzyme that creates the new DNA strand by joining the recipient and donor DNA through bonds
What is transformation and what are transformants?
If and When the phenotype of a bacteria’s genome is changed by intake of donor DNA from the environment, it is called TRANSFORMATION; the progeny of the altered bacteria that express this change are called the TRANSFORMANTS.
What is a selfish genetic element?
A sequence of nucleotides that uses cells to reproduce and spread between them.
What are three differences between viruses and cells?
1 - Viruses do not have ribosomes and can't produce the proteins needed to reproduce independently
2 - They can use both DNA and RNA as their genetic material
3 - They cannot make energy from the utilization of nutrients in the environment; they must use their host's energy to reproduce.
What is a virus called when it is not in a host cell?
A particle or Virion.
What is a phage particle composed of?
a protein/lipid coat with a genome inside
What is lytic infection?
The general life cycle of a lytic virus, from infection of the host cell, hijacking of the host cells mechanisms, use of the host cells mechanisms to produce viral components, and assembly of the components into progeny viruses, to the destruction of the host cell by rupturing its plasma membrane (lysis) and the release of the progeny viruses so that they can spread and infect other cells.
What are the proteins needed by the phage to complete the lytic cycle?
Enzymes to break apart host DNA, catalyze assembly, lyse the host cell, and proteins to build the structure of the phage - capsid, tail and tail fibers.
What are the two ways a lysogenic phage can exist in a bacterial host cell?
- as a seperate plasmid-like DNA fragment
- as a part of the circular DNA chromosome of the bacteria...integrated through recombination
What's the difference between temperate and virulent phages?
Termperate can exist in either lysogenic or lytic form, while virulent can only be lytic.
What does lysogeny mean?
a dormant state in which a bacterial phage exists in a bacteria cell as part of the host cell’s DNA
What does the term prophage refer to?
A phage that is dormant (lysogenic) inside of a bacterial host cell.
What part do regulatory proteins play in the lysogenic cycle?
They maintain the state of the phage and keep it dormant.
What is phage conversion?
When some of the viral genes of a lysogenic phage are expressed with the bacterial genes and cause a change in the bacteria's phenotype.
How big are plasmids?
between 2 and 200 kilobase pairs
What are the three types of plasmids?
Resistance: code for proteins that give the bacterium resistance to antibiotics.

Nutritional: codes for proteins that allow the bacterium to use special molecules as nutrients.

Infection: codes for proteins that help the bacteria infect other organisms.
How are plasmids like viruses?
Plasmids act like viruses towards bacterial cells in that they also ‘infect’ them.
What is the major difference between viruses and plasmids?
Plasmids cannot exist in extracellular form like viruses...they are only passed from inside one cell to another.
What is conjugation?
when a male bacterium passes some of its genetic information into a female bacterium, which then incorporates it into its DNA and passes it on its progeny

The cell that has the plasmid is called the host cell and the donor cell, while the cell receiving the plasmid is called the recipient.
What is a conjugative plasmid?
A plasmid containing tra genes that code for Tra proteins, which mediate the transfer of the plasmid's DNA into another cell. Any type of plasmid can also be conjugative.
What is a Pilin and a Pilus?
A pilin is a specific protein coded for by the tra operon that forms (with many other copies) the pilus.

The pilus is an extension of the donor cell shaped like a tube that is used to make contact with the recipient cell and pull it in.
What does the pilus interact with and what encodes it?
The pilus connects to a receptor protein that is coded for by the bacteria's chromosomal DNA.
What is a conjugation bridge?
A fusion of two bacterial membranes resulting from the reception of the recipient cell of the donor's pilus (used to draw the cells together).

The bridge is what the transferred genetic information travels through.
What is Nickase and what area of a bacterial plasmid does it interact with during conjugation?
Nickase is an endonuclease enzyme that breaks the plasmid's DNA strands apart.

This happens at the origin of transfer (oriT) sequence, which is near the tra operon.
How does replication of the other plasmid DNA strand occur?
It happens at the same time the strand is imported into the recipient cell - in both cells. Each ends up with a semiconservative DNA strand.
How fast can the transfer of a 100 kilobase pair plasmid occur?
In 2 minutes.
What is the effect of conjugation on:
1) The donor cell
2) The recipient cell
3) The plasmid
The donor cell recieve no change.

The recipient cell is affected if it gains any new characteristics coded for by the plasmid.

The plasmid is able to spread itself around because it always conserves one copy and transfers one copy.
What is the cause of a cell's loss of its plasmid? (specific mechanism)
The segregation system (protein-based partioning system). It is responsible for correctly dividing the plasmid copies into the daughter cells. Sometimes it makes an error and a daughter cell doesn't recieve a copy.
What is the advantage of a bacteria cell's cure of its plasmid?
It can replicate and divide faster without it, which gives it a selective advantage compared to its plasmid burdoned form.
What is a disadvantage of a bacteria cell's loss of its plasmid?
Some plasmids will kill the host cell if it is lost; a poisonous molecule is released.
What is transposition and what is a transposon?
A TRANSPOSON is a gene/gene set that can move from one location to another within a cell⬦this translocation is called TRANSPOSITION.
Transposition can involve the transfer of genetic information from a plasmid to the chromosome within the cell, OR from any region of DNA to another within the cell.
(2 locations within SAME DNA molecule, or 2 locations between locations on DIFFERENT DNA molecules).
What are the three compononets of a transposon?
1)a gene coding for a transposase protein involved in mediating the transposition process

2)Other genes that code for various characteristics (like antibiotic resistence)

3) Inverted Repeat Sequences (palindromes) that comprise the ends of the transposon
What is a target site (transposition)?
The location where a transposon is moved to in the recipient DNA. It is very short (5bp) and occurs randomely in multiple locations throughout the chromosome.
What are the four steps of transposition?
1) The transposon copies its target site...becomes the Inverted Repeats

2)Transposase binds to the transposon and excises it from the donor DNA at the ends of the IR's

3)Transposase (still attached) binds to the target site on the recipient DNA. It breaks the backbone there and inserts the transposon...each end is attached by a single stranded section

4)RNA polymerase fills in the ss gaps by synthesizing complementary nt's
What is a plasmid that has a commensalistic relationship with bacteria called?
cryptic plasmid
What kind of relationships can viruses have with bacteria?
They can be parasitic, mutualistic or commensalistic.
Why are viruses able to be less protective of their host cell (in comparison to plasmids)?
Because they can exist extracellularly...if released through a cell's death they are protected by their protein coat. The plasmid is vulnerable outside of the cell.
What kind of relationships can transposons have with bacteria?
They can be either mutualistic or commensals.
What type of transposon is considered to be a commensal?
Insertion Sequences (IS).
How do bacteria protect themselves from invading foreign DNA?
With a restriction-modification system
What is modification?
A base in a special recognition sequence is methylated by a special enzyme within a bacteria called Methylase. This marks it as host DNA and protects it from being destroyed by the restriction endonuclease.
What is restriction?
It is the selective destruction of DNA within a bacterial cell that is not specially marked, or modified (identifying it as host DNA). It happens at a special recognition sequence. Restriction endonuclease breaks the backbone of unmodified DNA.
How can an invading DNA escape restriction?
1) If it stays in cell long enough it will be modified by methylase

2)If it doesn't contain the recognition sequence marking it for destruction.
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