Bacterial Genetics Flashcards

1
Q

T or F. Bacteria encode a single RNA polymerase responsible for transcribing all needed RNAs

A

T

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2
Q

What is the region upstream of every coding sequence and is that spot for RNA polymerase binding?

A

the promoter region

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3
Q

What is an operator?

A

generally located near the promoter, and is a sequence that binds proteins involved in the regulation of the gene’s expression

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4
Q

One significant difference between eukaryotic and prokaryotic genes is the ________.

A

lack of introns in prokaryotic coding sequences.This feature, plus the lack of a nucleus in bacteria, allows for the coupling of transcription and translation in bacteria: translation of mRNAs begins before transcription of the message is completed.

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5
Q

What is an operon?

A

a cluster of genes, typically involved in a related function, that are transcribed on a single mRNA (i.e., a polycistronic message where cistron=gene), and are then translated independently to produce the individual proteins.

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6
Q

What is the lac operon?

A

a cluster of genes involved in the utilization of lactose by the Gram negative bacillus Escherichia coli.Thus, in the case of the lac operon, there is a single promoter and operator upstream of the first gene, lacZ, and a single transcription termination site downstream of the last gene, lacA. All three units (lacZ, lacY, and lacA) contain their own ribosomal binding sites to independently translate their portion of the mRNA to produce the corresponding proteins

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7
Q

What are the corresponding proteins of lacZ, Y, and A on the alf operon of E. coli?

A

B-galactosidase,lactose permease, and lactose transacetylase, respectively.

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8
Q

How is the translation of proteins in bacteria different from that in viruses?

A

The translation of individual proteins from a polycistronic RNA template in bacteria can be contrasted with a process seen in certain RNA viruses of animals where a polycistronic RNA template is translated into a single “polyprotein” that is then cleaved into individual proteins

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9
Q

The clustering of functionally-related genes into operons serves at least two purposes. What are they?

A

(i) operons help to ward off loss of function because if genes contributing to a single process reside in different regions of the genome, they can be more easily lost to evolutionary divergence, and (ii) the use of a single promoter and operator provides co-regulation of the genes in the operon, facilitating coordinate expression of functionally-related genes.

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10
Q

What are the three types of gene regulation in bacteria?

A

(i) constitutive, where there is effectively no regulation, the genes are always expressed, (ii) positive regulation, in which an activator protein promotes RNA polymerase binding to promoters and therefore facilitates expression, and (iii) negative regulation, in which a repressor protein binds the operator sequence and prevents transcription by RNA polymerase until the repressor is removed.NOTE: Because transcription and translation expend energy, constitutive expression is rare; most wild type genes are regulated.

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11
Q

What do inducers do?

A

Regulation is often provided through the use of inducers, molecules that either interact with activator proteins, causing them to bind at operators and promote RNA polymerase binding at nearby promoters, or interact with repressor proteins, preventing them from binding operators and subsequently blocking transcription by RNA polymerase.

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12
Q

T or F. Negative regulation appears to occur less often than positive regulation.

A

F. Negative regulation appears to occur more often than positive regulation. If you consider that most mutations are deleterious, this makes sense: loss of a repressor allows continued gene expression, but loss of an activator leads to loss of gene expression, and hence, function.

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13
Q

What does the lacI gene do?

A

the lac operon and encodes a repressor protein; lacI is constitutively expressed.n the absence of lactose in the growth medium, the repressor protein is found predominantly bound to the lac operator, where it prevents RNA polymerase from transcribing the operon.This makes sense – in the absence of lactose, it would be wasteful, in energy terms, to transcribe and translate the lac proteins when the sugar substrate is unavailable. The energy expenditure would not be recouped.

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14
Q

What happens if lactose is present?

A

However, if lactose is present in the growth medium, catabolism of the lactose would lead to a net gain in energy for the bacteria. Therefore, in the presence of lactose, the sugar is transported inside the cell where it is converted to a related structure, allolactose, which serves as the inducer of lac expression

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15
Q

How does allolactose induce lac expression?

A

As with any bound ligand, the lacl repressor releases from the operator at low frequency, and when this occurs in the presence of the inducer allolactose, the allolactose binds the repressor protein, altering its conformation, an outcome termed allostery. The altered conformation prevents the repressor from binding the lac operator, thus freeing the operon from repression.

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16
Q

What happens once the lactose has been used up?

A

Once the lactose has been used up from the growth medium, allolactose will no longer be formed, lac repressor molecules will no longer be allosterically altered, and they will return to the operator site to again repress lac operon expression.

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17
Q

While operons are an efficient structure for co- regulation, functionally-related genes do not always cluster into a single operon. When independently transcribed genes, or multiple operons, are controlled by the same regulatory protein or process, they are considered to constitute a _____.

A

regulon

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18
Q

What is the two-component signaling pathway of bacteria?

A

named because of a sensor found in the cytoplasmic membrane and an intracellular transducer The binding of ligand to the sensor leads to the sensor’s phosphorylation and subsequently to the phosphorylation of the intracellular transducer. Once phosphorylated, the transducer is then able to function as an activator and bind the operator of genes whose expression will lead to an appropriate response to the extracellular ligand.

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19
Q

What is the advantage of the two-component signaling pathway?

A

This represents a way that bacteria can respond to changes in extracellular conditions without the need to transport the ligand inside the cell, as is the case in lac operon regulation. Changing temperature, osmolarity, or availability of extracellular iron are examples of conditions in which two-component regulatory systems are used by bacteria to respond appropriately.

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20
Q

How do bacteria communicate with each other?

A

by releasing specific signaling molecules and by measuring the local concentrations of signaling molecules in their environment. As the cell density of the population increases, the concentration of signaling molecules also increases.

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21
Q

What happens when a concentration threshold of the signaling molecule is reached?

A

the cells respond by turning on a new set of genes. This ability of the cell to sense the cell density is called quorum sensingSome bacterial pathogens use quorum sensing to coordinate the expression of virulence genes needed to escape the immune response or otherwise establish infection. Quorum sensing is especially important in biofilm formation.

22
Q

What is the composition of the signaling molecules used by bacteria?

A

The signaling molecules are not peptides, but rather tend to be cyclic structures such as lactones or quinolones.

23
Q

T or F. Most bacteria have circular genomes, and often this is contained on a single piece of DNA.

A

T

24
Q

What are the challenges of bacteria being haploid?

A

(i) mutations readily lead to loss-of-function since a second copy of the gene is not available to compensate, and (ii) phenotypic diversity could be limited in the absence of allelic pairs.bacteria overcome these limitations in part just be sheer numbers. In the case of spontaneous lethal mutations, the loss of a few bacteria among the millions present will hardly influence the outcome of an infection.

25
Q

As for phenotypic diversity, bacteria have evolved some low frequency genetic events that lead to new gene expression within a subset of the population, and subsequently, to selective amplification of the new phenotypic progeny under appropriate conditions. What are they?

A

phase variation and antigenic variation

26
Q

What is phase variation?

A

results in an “on/off” expression of a gene or subset of genes. The “genetic switch” that controls the “on/off” expression is often the simple inversion of a gene so that in the “on” position it is aligned with its promoter and expressed. In the “off” position, the coding sequence has inverted or “flipped” so that the promoter is no longer at the start of the gene.The “genetic switch” may encode a structural protein or a regulatory protein. For example, in uropathogenic E. coli, the most common cause of urinary tract infections, phase variation controls the expression of a regulatory protein that in turn regulates the expression of a particular pilus protein necessary for bladder colonization. The frequency of the switching is roughly 1 in every 1,000 bacteria.

27
Q

What is antigenic variation?

A

Antigenic variation results in the expression of a large array of surface components for some bacteria. Example: The process by which Neisseria gonorrheae potentially expresses 1,000s of different variations of surface pili is shown in Figure 3. Pili are produced from the pilE gene, where the “E” stands for “expressed.” That is, the pilE gene is aligned with its promoter and is transcribed and translated. The resulting proteins contain conserved and variable regions. The variable regions are derived from many pilS coding sequences, where the “S” stands for “silent.” The pilS sequences lack promoters, and are only expressed when recombined into the variable coding region of a pilE gene. This results in the expression of a new variant of the pilus protein. Because the pilus is a major antigen during neisserial infection, the process is termed antigenic variation, and the expression of a new pilus variant allows the bacteria to escape the immune response and reestablish infection. Antigenic variation occurs at a frequency of about 1 per 1,000 bacteria, so as the new variants are amplified in the face of an immune response to the previous variant, the clinical presentation is one of recurring infection.Note that unlike conventional gene regulation described for the lac operon or two-component signaling, only a subset of bacteria alter their gene expression under phase or antigenic variation. This provides phenotypic diversity to the population, but also requires large numbers of bacteria and rapid growth to impact the infection.

28
Q

T or F. A third consequence of the haploid nature of bacterial genomes (along with asexual reproduction) is that genetic diversity is not passed down to daughter cells through allelic segregation.

A

T

29
Q

While phase and antigenic variation can generate phenotypic diversity, other methods for generating genetic diversity are essential to the long-term survival of a species. Four methods of genetic exchange that promote the acquisition of new genes are:

A

(i) transposition, (ii) conjugation, (iii) transduction, and (iv) transformation.

30
Q

What are Transposons?

A

mobile genetic elements that are said to “hop” or transpose from one sight in a genome to another, sometimes leaving the original copy behind and thus incrementally increasing the number of transposons in the genome.

31
Q

What are the four basic components of transposons?

A

(i) insertion sequences at each end of the transposon that contain inverted repeats and allow for integration of the transposon into the target DNA, (ii) a transposase, a recombinase that executes the integration of the insertion sequences, (iii) a repressor protein that regulates the frequency of transposition, and (iv) a gene that benefits the host – usually an antibiotic resistance gene, but sometimes a toxin or other virulence factor. Because the fourth gene benefits the host, it helps to maintain transposons in a population of bacteria.

32
Q

Why is it important that transposons contain a gene that benefits the host?

A

This is significant because transposons actually function as mutagens inside of cells. Because of their size and complexity, they can disrupt normal transcription and translation when they insert within a gene or operonConsider the lac operon once again: if a transposon inserted in the lacZ gene, not only would it prevent production of B-galactosidase, but it would also stop transcription of the downstream lacY and lacA genes, thus preventing the production of the permease and transacetylase. In this regard, transposons can introduce genetic diversity in two ways: (i) introduction of an antibiotic resistance gene or some other new gene, and (ii) creation of mutants lacking nonessential wild type functions.

33
Q

Transposons are an important source for creating antibiotic resistance among bacteria, but recognize that transposition is an intracellular process – transposons cannot directly “hop” from one bacterium to another.

A

Transposons are an important source for creating antibiotic resistance among bacteria, but recognize that transposition is an intracellular process – transposons cannot directly “hop” from one bacterium to another.

34
Q

The introduction of a transposon from one cell to another relies on what process?

A

conjugation, a mating process that can occur between similar, but not necessarily the same, species of bacteria.

35
Q

What are plasmids?

A

Plasmids are extrachromosomal (i.e., episomal), circular DNAs found in most bacteria. Plasmids are double-stranded DNAs, and range in size from 2-500 kilobase pairs.

36
Q

Does plasmid replication occur always with DNA replication within a bacteria?

A

Plasmid replication can be directly tied to bacterial genome replication, but some smaller plasmids can also replicate independently of the genome to amplify their copy number within the cell, sometimes to greater than 25 copies per cell.

37
Q

T or F. Plasmids are major targets of transposons within a cell

A

T. Within a cell, plasmids are targets for transposons, and because plasmids do not possess genes essential for cell survival, the antibiotic resistance genes that transposons impart to plasmids can be one way that plasmids are maintained within a population of bacteria.In fact, resistance to increased concentrations of an antibiotic by a particular bacterium can sometimes be the result of amplification of a plasmid harboring a transposon encoding the resistance.

38
Q

What must a donor bacterium have in order to for successful conjugation to occur?

A

donor bacterium must contain an F (fertility) factor, a plasmid that carries the genes necessary for its transfer to a recipient cell.If it has this, The donor is said to be “F+” ormale. Significantly, the F factor encodes for the sex pilus that is necessary to establish direct contact with a recipient bacterium, which lacks the F factor (“F-“).

39
Q

How does conjugation occur?

A

Once direct contact is established, the circular F factor is cleaved so that one strand crosses the bridge between the two bacteria and enters the recipient cell. Once there, second strand synthesis occurs along with circularization so that the recipient now contains a copy of the F factor (the original donor bacterium remains F+). As a result, the recipient is now male (F+) and capable of initiating conjugation.

40
Q

Transfer of a single F factor from one cell to another does not by itself create great genetic diversity. How does it improve diversity then?

A

F factors can at times facilitate the conjugation of other, non-mobile (i.e., nonconjugative) plasmids from an F+ host to a recipient, and perhaps more significantly, an F factor can transfer parts of the donor genome to the recipient

41
Q

How can transfer of parts of the donor genome occur?

A

Transfer of parts of the donor genome occurs only after the F factor has first integrated into the bacterial chromosome. Once the F factor initiates transfer in this circumstance, the F factor transfers trailing chromosomal genes that lie downstream of the F factor integration site until the sex pilus bridge is disrupted.The newly introduced chromosomal genes can then be stably recombined into the recipient genome, significantly altering its genetic diversity. Because this is a relatively efficient way to transfer large amounts of DNA from one bacterium to another, the cells containing the integrated F factor are called Hfr cells, for high frequency transfer.

42
Q

Hfr transfer is believed to be a major mechanism for the generation _______ in pathogenic bacteria.

A

of pathogenicity-associated islands (PAIs)

43
Q

What are PAIs?

A

large regions of a chromosome that encode various virulence factors such as type III secretion systems, toxins, and iron acquisition components. Significantly, non-pathogenic strains of the same bacterium lack PAIs

44
Q

By definition, F factors do not encode antibiotic resistance genes. If a plasmid acquires an antibiotic resistance gene, most likely by acquiring a transposon as discussed above, it is termed an _____

A

R-plasmid or R- factor (“R” for resistance)R-factors can be conjugative or dependent on F factors for conjugation, but they are a major reason for the spread of antibiotic resistance because they can be transferred across species, and at lower rates, sometimes across genera. Moreover, R- factors tend to accumulate multiple antibiotic resistance genes and therefore encode multidrug resistance.

45
Q

Viruses that prey on bacteria are termed _____.

A

bacteriophage, or phage for short

46
Q

What are the two general categories of phage?

A

i) lytic, in which the phage infect, replicate in large numbers, and then are released from cells, usually by cell lysis, so that they may infect other cells, and (ii) temperate (or lysogenic), in which phage infect cells, but instead of undergoing lytic replication in all cells, the phage genomes integrate at specific sites in the bacterial chromosome of some cells. The integrated phage genome is known as a lysogen, and the lysogen replicates as part of the bacterial chromosome until some form of stress to the cell induces the lysogen to excise from the chromosome and undergo lytic replication. The cycle is then repeated.

47
Q

Some lytic and temperate phage can “inadvertently” transfer bacterial genes from a host to a subsequently infected cell. The transfer of genetic material from one bacterial cell to another via phage is termed _____.

A

transduction.

48
Q

What are the two forms of transduction?

A

generalized and specialized.

49
Q

What is generalized transduction?

A

Generalized transduction is carried out by strictly lytic phage only. As part of their strategy to overtake a bacterial cell and use it for phage replication, lytic phage cleave the bacterial chromosome into small fragments. These fragments tend to be the same size as the virus genome and can be mistakenly inserted (or “packaged”) into phage particles instead of the phage genome during replication, at a frequency of about 1 per 1,000 particles.The mis-packaged phage particles can mimic a normal infection and insert the bacterial chromosome fragment into a recipient cell, where the fragment can be stably recombined into the new bacterial genome. Because any bacterial chromosomal segment can be mis-packaged, any bacterial gene can be transduced to the recipient cell (but just once). Hence, the term generalized transduction.

50
Q

What is specialized transduction?

A

Specialized transduction is carried out by temperate phage, and is far more important than generalized transduction in terms of providing genetic diversity, and more specifically, virulence factors to recipient bacteria. This lysogeny occurs at specialized sites in the chromosome, and normally, the lysogen would excise precisely before entering lytic replication. However, the phage will infrequently (on the order of generalized transduction) undergo an aberrant excision and mistakenly incorporate an adjacent bacterial gene into the phage particle. This can result in a new, stable form of the temperate phage, and subsequent infection and lysogeny will always transduce the acquired bacterial gene to recipient cells. Because the original lysogen integrates at a specific site in the bacterial chromosome, only a few specific bacterial genes can be transduced by a particular temperate phage, hence the term specialized transduction. Temperate phage have evolved over time to harbor important virulence factors; for example, cholera toxin, which leads to profuse diarrhea, is contained on a temperate phage, and only Vibrio cholerae species that are lysogenized by the phage cause significant disease.

51
Q

What is transformation?

A

Transformation is the uptake of naked DNA from one cell to another. While this is an important laboratory procedure in the fields of molecular biology and gene cloning, it likely plays an insignificant role in creating genetic diversity in nature. If a bacterial cell is able to take up DNA from its surroundings, it is said to be competent. Molecular biologists routinely manipulate bacteria to make them competent in the laboratory. However, very few pathogenic bacteria have been shown to be “naturally” competent. An exception is Streptococcus pneumoniae

52
Q

Process% Bacterial Chromosome TransferredConjugation25-50%Transduction1-2%Transformation5-10%

A

Process% Bacterial Chromosome TransferredConjugation25-50%Transduction1-2%Transformation5-10%