Chapter 9 - Bacterial Genetic Analysis

The notation uvrB- indicates

the bacteria has a mutated copy of this gene

Bacteria are excellent model organisms for the study of genetics for all the following reasons except

they have a relatively small, diploid genome that is easier to characterize.

A property common to all restriction sites is:

they identify where both strands of the DNA double helix should be cut.

A common strategy to reduce the probability of cells developing spontaneous mutations (reversions) in nutritional mating experiments is to

use strains that are that have more than one mutation.

Mutations may result is

loss of gene function. restoration of gene function lost previously. modification of gene function.

The lambda phage genome is circular with 7 EcoRI restriction sites. When digested and separated on an agarose gel, how many bands (restriction fragments) should appear?

7

What is a shuttle vector?

A shuttle vector is a plasmid that can replicate in two unrelated types of cells (e.g. E. coli and Yeast). These plasmids are designed so that they have two origins of replication - one that works in one type of cell and a second origin that works in the other. Typically these shuttle vectors also contain multiple selectable markers that enable selections in both types of cells.

What are transposons?

A transposon is a sequence of DNA that can change its relative position within the genome of a single cell. The mechanism of transposition can be either replicative ("copy and paste") or non-replicative ("cut and paste"). Insertion sequences, or IS elements, are variants of transposons that encode only the proteins (i.e. transposases) and inverted repeat sequences necessary for their own movement to a new location. Transposons are more complex than IS elements in that they encode additional genes not involved in transposition including, sometimes, antibiotic resistance genes.

What is a wild-type strain?

A wild-type strain usually possesses the typical or representative characteristics of the species. In many cases these strains were isolated directly from a natural source but in some instances the term is used to describe a commonly used laboratory strain from which all mutant strains in a study have been derived.

What are bacterial nutritional mutants, or auxotrophs? Give an example.

Auxotrophs are strains of bacteria that have lost the ability to synthesize a specific essential molecule (e.g. an amino acid like histidine). Therefore these strains must be grown in media that contains the essential component. For example a histidine auxotroph must be grown on media that contains histidine.

Describe how mutant genes and proteins are named.

Bacterial genes are named with a three letter, lowercase, italicized identifier. If there is more than one gene involved in a particular function then a single uppercase italicized letter is appended to the end. For example lacZ, lacY and lacI are three different genes involved in the utilization of lactose in E. coli. The corresponding proteins encoded by the lacZ, lacY and lacI genes are named LacZ, LacY and LacI; for proteins the first letter is capitalized and the name is not in italics. If a particular gene is mutated such that it loses function in a particular strain a superscripted minus symbol can be used. For example an E. coli strain that carries a large deletion in the lacZ gene can be called a "lacZ- mutant strain"

How do the genome arrangements of bacterial and eukaryal cells differ?

Bacterial genomes are smaller and more compact (encode more proteins per unit of DNA) than eukaryal genomes. The chromosomes of eukarya are linear whereas in bacteria they are typically (but not always) circular . Bacteria also frequently harbor smaller genetic elements such as plasmids, whereas they are rare in eukarya.

A bacterial genome typically is composed of what types of molecules?

Bacterial genomes are typically composed of chromosomes and plasmids, each of which can vary in number. Many bacteria contain a single circular chromosome, but some bacteria have linear chromosomes or multiple chromosomes. Bacteria can also harbor several different types of plasmids, the numbers of each can vary between one and several hundred per cell.

What is copy number, and how is it controlled in the bacterial cell?

Copy number refers to the amount of a given plasmid present in a bacterial cell. Some plasmids exist in multiple copies whereas others are present at only one copy per cell. Copy number of a plasmid is typically controlled by RNA or proteins encoded on the plasmid that regulate the initiation of DNA replication.

What are cosmids?

Cosmids are plasmid vectors that contain cos sites, which allow the plasmid DNA to be packaged into a phage particle, which can be done in vitro by mixing the vector with phage heads and phage tails. Because a phage can carry up to 50 kb of DNA it is possible to clone very large fragments into these vectors and easily get them into E. coli.

Create a table explaining F+, F−, Hfr, and F′.

Explanation F+ A strain of E. coli that harbors the F (fertility) plasmid. F− A strain of E. coli that lacks the F-plasmid. This strain is capable of receiving F-plasmid DNA by conjugation (at which point the cell would become F+) Hfr A "high frequency of recombination" strain. Hfr strains have a copy of the F-plasmid integrated into their chromosome. F' A strain of E. coli that harbors an F plasmid that carries genes from the E. coli chromosome. These F' plasmids are created in rare events when the F plasmid in an Hfr strain recombines back out of the chromosome and in the process carries a segment of E. coli DNA with it.

The difference between F+ and F- cells is

F+ cells contain the fertility plasmid and are able to initiate conjugation while F- cells do not have the fertility plasmid and cannot initiate conjugation.

T/F: Bacterial genomes consist of a single, circular chromosome

False

T/F: Mutations normally result in loss of function.

False

Describe the process of homologous recombination

Homologous recombination is the process by which two molecules with a stretch of identical (or nearly identical) sequence are joined. Homologous recombination initiates when single-stranded breaks are introduced into one of the DNA molecules. The RecA protein binds to the single-stranded DNA that results from this nick and searches the other DNA duplex for regions of homology while displacing one of the strands at that region. A reciprocal reaction occurs with the other strand to generate a four-stranded cross-shaped DNA structure known as a Holliday junction. The Holliday junction is subsequently cleaved and the end product of the reaction is two recombined DNA molecules.

T/F: Transformation involves the uptake of free pieces of external DNA while conjugation involves the transfer of specific pieces of DNA through cell-to-cell contact.

True

What advantages do phage vectors have?

Phage vectors enable the cloning of large inserts of DNA into the middle of an active phage (typically phage lambda). This DNA can be packaged into a phage particle in vitro, and then used to infect bacteria. The newly created phage, which has been engineered such that it cannot integrate into the host chromosome, can be grown on bacteria to make large quantities of the desired DNA. The amount of DNA that can be cloned into vectors derived from phage lambda can be up to 20 kb.

What is replica plating, and how was it used to show that antibiotic-resistant mutants arise in the absence of selection pressure?

Replica plating is when a sterile cloth pad is stamped onto a "master plate" containing colonies of bacteria. The cloth pad containing bacteria from the master plate is then applied to a "replica plate" to transfer bacteria (only a fraction of the bacteria from the master plate are picked up by the cloth). In this way, the replica plate has identical types of bacterial colonies in the same position as the colonies on the master plate. This enables a researcher to test many colonies at the same time for multiple phenotypes, for example by copying the master plate onto plates containing different antibiotics or nutrient conditions. By looking at which colonies grow (or fail to grow) on the replica plate the researcher can look for bacterial colonies that fail to grow under a particular condition. This approach was used by Esther Lederberg to show that bacteria could spontaneously become resistant to the antibiotic streptomycin even in the absence of the drug. She made a master plate of wild-type bacteria that were grown without streptomycin. She then made a replica of this master plate where the agar contained streptomycin and found that she could obtain several streptomycin-resistant colonies. Finally she could show that the same areas with streptomycin-resistant colonies on the replica plate, also harbored resistant bacteria on the master plate whereas areas of the plates that gave no colonies on the replica plate harbored no resistant bacteria on the master plate. This convincingly demonstrated that the master plate had streptomycin-resistant bacteria even though those bacteria had never been exposed to the drug.

Which of the following is true about bacterial plasmids?

Replicate independent of the chromosome

Describe the activity of restriction enzymes

Restriction enzymes are proteins that cleave DNA at specific sequences. For example the restriction enzyme EcoRI cuts DNA at the sequence 5'-GAATTC-3'.

Explain blue-white screening.

Sometimes, when cloning into a plasmid, only a small fraction of the plasmids actually contain the desired fragment while the remainder of the plasmids re-ligated with no insert. Using blue-white screening a scientist can determine visually if the colony of bacteria harbors a plasmid with an inserted fragment of DNA. The plasmid is designed such that the cloning site lies in or just upstream of a gene encoding the N-terminal (α) domain of beta-galactosidase. The E. coli strain that is used for the cloning procedure expresses the C-terminal (ω) fragment of beta-galactosidase. Colonies of E. coli harboring plasmids that do not contain the cloned DNA fragment will express both the α and ω fragments, which combine to make a functional beta-galactosidase enzyme. When these cells are grown on agar containing the compound X-gal they turn blue due to the activity of the beta-galactosidase enzyme. Plasmids that have successfully obtained an insert of DNA will not make a functional α fragment because the gene encoding the α fragment is interrupted. Therefore colonies of E. coli that harbor plasmids containing an insert will remain white on agar containing X-gal.

The difference between Hfr and F+ strains is

The fertility plasmid is integrated into the host cell chromosomal DNA in Hfr cells while the fertility plasmid is independent in F+ cells

What is transduction? Explain the role of bacteriophage in the process.

Transduction is the transfer of DNA from one cell to another via a phage particle. This process occurs when some phage progeny accidentally package host DNA instead of phage DNA. Phage particles that carry host DNA are called "transducing particles". These transducing particles are essential DNA injection machines that can carry their DNA to a new host but, because they don't carry phage DNA, will not kill the recipient cell. DNA that is injected into the new cell can integrate into the host chromosome.

Which of the following is not a mechanism that moves DNA between bacteria?

Transformation

What is DNA transformation, and how are competent cells involved in the process?

Transformation is the transfer of free DNA molecules from the outside environment directly into an organism. Some bacterial species are naturally competent and encode specialized DNA receptors and uptake systems that enable them to import DNA across their membranes. Other bacterial species, like E. coli, do not encode such systems and cannot take up DNA naturally. They will only take up DNA if the cell is manipulated in the laboratory using techniques like calcium treatment or electroporation.

T/F: A cosmid is a hybrid plasmid/phage vector that combines beneficial cloning properties of both plasmids and phages.

True

T/F: Bacterial genomes include both chromosomal and plasmid DNA

True

T/F: Competent cells are those able to take up pieces of foreign DNA from the environment.

True

T/F: Only restriction enzymes that yield short regions of single stranded DNA (sticky ends) are useful for generating recombinant molecules in precise and predictable ways.

True

T/F: The term mutant refers to a cell or strain and the term mutation refers to a change in DNA sequence relative to the wild type.

True

Describe what is meant by plasmid incompatibility.

Two different plasmids are incompatible if they cannot exist stably within a population of cells. This occurs when two plasmids share very similar mechanisms to control their replication and copy number. During replication of two incompatible plasmids, the cell cannot distinguish how many copies of each plasmid it has since both plasmids utilize the same method of counting (to the cell they look the same). If one plasmid is copied in excess of the other it will dominate the population of plasmids in the daughter cells. After the next round of replication, the bias toward one plasmid will be even greater due to the fact that the more abundant plasmid has more chances to be copied than the less abundant plasmid. Within a few cell divisions the less abundant plasmid can drop to numbers so low that one of the daughter cells will inherit no copies and hence the plasmid is lost.

Distinguish between horizontal gene transfer and vertical gene transfer.

Vertical gene transfer is the inheritance of genetic material from a direct ancestor (e.g. mother cell to daughter cell). Horizontal gene transfer is the transfer of genetic material between microbes (e.g. between E. coli and Salmonella).

Explain the processes of selection and screening.

When a scientist searches for a new mutant strain of bacteria with a desired phenotype there are two general approaches that can be employed. In some cases it is possible to conduct a selection experiment. In this type of experiment conditions are chosen such that a mutant strains grow much better than wild-type strains. An example of a selection is during the identification of mutant bacteria that resist antibiotics. In such an experiment a culture of bacteria is spread on nutrient agar supplemented with a certain concentration of antibiotic. Mutants that can resist the antibiotic grow while the wild-type bacteria are killed off. The advantage of selections is that millions of bacteria can rapidly be assessed for the presence of a mutation. When hunting for a particular mutant using a screening experiment the wild-type and mutant bacteria grow at similar rates under the condition of interest. The scientist must look at thousands of colonies to find the ones with a property of interest. This is often done by looking for changes in colony color or morphology. Using a screen to find a mutant with a desired phenotype is much more laborious than using a selection because of the high background of wild-type colonies compared to the relatively few mutant colonies and the fact that each colony must be examined individually

What purpose does a selectable marker play in a plasmid vector?

When cloning a fragment of DNA into a plasmid using restriction enzymes and DNA ligase there are a variety of products that occur. Not all of the restriction enzyme cut plasmid re-closes to generate a circular plasmid. Furthermore, although there are various methods for getting DNA into a bacterial cell, these processes are very inefficient and some of the bacteria successfully take up DNA. Furthermore many cells will take up DNA that is linear plasmid or a fragment of DNA that will not replicate. By using a plasmid containing a selectable marker (such as resistance to ampicillin) only cells that have taken up a closed circular plasmid with an origin of replication will be able to grow under the selective condition (for example, on media containing the antibiotic ampicillin).

Distinguish between single- and double-crossover events. How are they used for genetic manipulation in bacteria?

When two molecules of DNA recombine in a single crossover event they form a single molecule where a complete copy of one DNA molecule inserts into a single site on a second DNA molecule - such a molecule is called a "cointegrate". Double crossovers occur when two recombination events happen at different sites between two DNA molecules, causing an exchange of a segment of DNA between the two molecules. Both single and double crossover events can be used to manipulate bacterial genomes. Using a "suicide plasmid" unable to replicate in a specific host it is possible to select for single crossover events. The plasmid would carry a segment of DNA that is homologous to a gene of interest on the bacterial chromosome and a selectable marker (antibiotic resistance, for example). Single crossover events result in a cointegrate where the gene of interest is interrupted by sequence from the plasmid. These rare events can be selected by plating the cells on media containing antibiotic. Cointegrates generated by single crossover events are unstable over time since they carry homologous DNA on either end of the inserted molecule, which means that the cointegrated suicide plasmid can recombine to jump back out of the chromosome. Double crossover events can be used to replace a segment of DNA on the chromosome with a different segment of DNA. These events are difficult to obtain since they involve two crossover events instead of just one, but because the resulting product lacks homology at its ends it is much more stable than cointegrates generated by single crossovers. Plasmids used to select for double cross-over events carry two selectable markers - one that positively selects for replacement of the chromosomal gene (typically an antibiotic resistance cassette that is flanked by regions of homology to the gene the researcher wishes to delete) and a second marker that can select for loss of the plasmid (typically the sacB gene, which kills bacteria in the presence of sucrose). By selecting for cells that are resistant to both sucrose and antibiotic it is possible to find those cells where the gene of interest was replaced by the antibiotic resistance gene and lost the remaining plasmid sequence.

A desirable property for a good cloning vector is:

a piece of DNA that is able to replicate autonomously within a bacterial cell. a piece of DNA that has been well characterized including known restriction sites. a piece of DNA that includes easily detectable marker genes.

A wild type strain is

a strain that possesses typical or representative characteristics of the species.