Antibiotic Resistance

What is the difference between what phages and plasmids in terms of transfer between hosts?

Phages: very SPECIFIC - usually transfer between hosts of same or very closely related species Plasmids: can be very NONSPECIFIC - transfer between hosts that are very different from each other

The process of transformation involves what? What does "competence" mean and how is it obtained?

Transformation involves the release of naked DNA into the environment by the lysis of some cells (the donors), followed by the uptake of that DNA by other cells (the recipients). The ability to take up DNA from the environment is called competence. In many species of bacteria, competence is encoded by chromosomal genes. These genes become active under certain environmental conditions. In such species, transformation occurs readily: it is natural.

How do transposable elements move? Do they move between cells or within them? What ability does transposition rely on? What is their clinical significance?

Transposable elements mediate self-transposition & jump from genome to genome WITHIN THE SAME CELL Transposition relies on the ability of these transposable elements to synthesize their own specific recombination enzyme. Clinical Relevance of Transposition. Transposons are a major carrier of antibiotic resistance genes (evolution of R plasmids). -- Since they can jump from one genome to another, they can find their way to phage and conjugal plasmids. --In this manner, they can participate in the general transfer of genetic material from cell to cell and from species to species.

How does transposition of insertion sequences occur? Is it a common event?

Transposition occurs INFREQUENTLY. It involves recognition by the recombinase of the terminal inverted repeats and a target area into which a new copy of the IS element will be inserted. Here, I have shown an IS element located within a chromosome. Below it is a plasmid. After transposition into the target area, the original copy remains at its original site.

Give 2 examples of virulence factors carried by a phage

--Beta - toxin produced by Corynebacteria diptheria Beta - phage --Cholera toxin produced by Vibrio cholerae CTX phage*

If you knew gene exchange was occuring, what experiment could you do to distinguish between transformation and conjugation?

1) Add detergents or nucleases to environment, if it gene exchange stops --> it is transformation If gene exchange is uninterrupted --> can't be transformation 2) Vortex donor and recipient DNA together --> if gene exchange stops, it is likely conjugation (Sensitive to sheering, but not nucleases/detergents)

What type of recombination occurs in transformation?

homologous recombination

The answer to maintaining long-term effective use of therapeutic agents lies in what?

in better, more prudent use of antibiotics in human and animal health care

What is the range in size of genomes? Is it true that genome size is exactly correlated with complexity?

10^4 bp (small viruses) vs. 10^12 bp (amoeba) No - amoeba's genomes are larger than humans'! (~10^9)

The bacterial genome is called what else? The plasmid genome is called what else? The bacterial virus genome is called what else?

Chromosomal (Bacterial) Genome Plasmid (Episome) Genome Bacterial Virus (Phage) Genome

What type of plasmids are F and R plasmids?

Conjugative (or conjugal) plasmids

How does the bacterial genome change depending on its level of transcriptional activity?

In the late stationary phase/large phase, DNA is TIGHTLY PACKED and thus TRANSCRIPTIONALLY INACTIVE In the early stationary phase, DNA becomes somewhat looser (supercoiled loop/coral reef) In the exponential growth state, DNA is now LOOSELY arranged and transcription is ACTIVE

To transposons transfer DNA between cells?

NO! Within the same cell!!!!!!

What is the difference between an R and F plasmid?

R = resistance, F = fertility R plasmids carry genes for antibiotic resistance, F plasmids do not.

What determines the name of a transposon? If it carries antibiotic resistance genes, what is it called? If it carries genes for conjugation? If it carries a bacteriophage?

The genes between the insertion sequences 1) antibiotic resistance = transposon 2) Genes for conjugation = conjugal transposon 3) bacteriophage = tarnsposable phage

The chromsomal genome: -Large or small? -Single or double stranded DNA? -Tightly or loosely packed? -haploid or diploid? -circular or linear? Describe the most typical chromsomal genome.

large (megabases - btwn 2000-6000 genes, average 4000 genes) double-stranded DNA tightly packed structure (nucleoid) Typically, but not always, haploid can be either - circular or linear Typical chromsomal genome: single/haploid chromsome composed of one large (% mill bases) circular molecule of double stranded DNA.

Explain the mechanism of transformation using Strep. pneumoniae as an example. What is the DNA particularly susceptible to and why?

1) By lysis, trauma, or death, donor cells release naked DNA into the environment. Donor enzymes chop the released DNA into smaller pieces. Since the DNA is naked, it is highly susceptible to environment factors, including detergents that inhibit binding and nucleases that destroy DNA. 2) Pneumococcal cells secrete a protein competence factor that induces cells to synthesize special proteins necessary for competence so that chromsome of host becomes competent. 3) A protein located on the cell surface binds double-stranded DNA indiscriminately. It binds any DNA present in the environment. 4) As the DNA passes through the membrane, one strand is degraded by a NUCLEASE. Other proteins protect the DNA so it does not get degraded in the cytoplasm. 5) Remaining strand has 3 fates: degradation, circularization, recombination

Why is conjugation often called "mating?" Donor DNA is transferred to the recipient in the form of what? What is the recipient called once it has received the plasmid? What is DNA sensitive to?

1) Conjugation involves cell-cell contact, therefore it is sometimes called mating. 2) A CONJUGAL PLASMID: The donor cell contains a plasmid (an autonomously replicating, extrachromosomal molecule of circular double-stranded DNA). --Because all the genes required for conjugation reside on this plasmid, it is called a conjugal plasmid. --The recipient does not carry the conjugal plasmid. 3) Once the recipient has received the plasmid, it is called the exconjugant. 4) SENSITIVE TO SHEERING, but NOT NUCLEASES OR DETERGANTS (Because DNA transfer occurs through cell-cell contact, DNA is never naked and, thus, is not exposed to nucleases or detergents) 5) Once the recipient has received the plasmid, it is called the exconjugant.

Plasmids that cause antibiotic resistance what? Most plasmids used for genetic engineering are what?

1) Conjugative 2) nonconjugative

Which plasmids can autonomously transfer between hosts? Which cannot?

1) Conjugative plasmids CAN, non-conjugative plasmids CANNOT on their own but can via a conugative plasmid

What components are necessary for transformation to occur?

1) DNA binding protein on the surface of the recipient cell 2) Nucleases to cleave one of two strands of the double stranded DNA 3) Transport system to bring DNA into recipient cell 4) Competence -specific single-stranded DNA binding protein: protects single stranded DNA so it is not chewed up in the cytoplasm 5) A COMPETENT chromosome that can take up the new material (some cells secrete competence factors that allow this)

What is another name for the plasmid genome? How does it replicate? What is its structure like? Is it large or small? circular or linear? What is its clinical relevance? Can a single cell have >1 plasmid? Do Gram + or -- bacteria have it?

1) Episome 2) autonomous, self-replicating - EASILY TRANSFERRED FROM HOST TO HOST 3) extrachromosomal, double-stranded DNA 4) tend to be small (kilobases) 5) usually circular, but can be linear 7) Plasmids often carry genes that encode virulence factors, e.g. pili or toxins, and proteins that confer antibiotic resistance ("R plasmids"), e.g. erythromycin or penicillin. 8) Single can can have >1 plasmid 9) Both gram + and gram - bacteria can have it

Which bacteria use use transformation to alter appearance of clinically important surface antigens --> permits evasion of host immune system?

1) Streptococcus pneumoniae (Gram +) 2) Haemophilus influenzae (gram -) 3) Neisseria gonorrhea (gram -)

What is a prophage? A cell that carries a prophage is called a what?

1) The lysogenic phage genome 2) lysogen

What are the 3 modes of genetic exchange? What is true of transfer in all 3? What is another name for donor and recipient DNA?

1) Transformation 2) Conjugation 3) Transduction All three involve a one-way transfer of DNA from a donor cell to a recipient cell. donor DNA = exogenote recipient DNA = endogenote

Are lytic viruses always lytic? Are lysogenic viruses alwas lysogenic?

1) YES 2) No - can become lytic

What is a transposable prophage?

A bacteriophage that is a transposon. As a phage, it can alternate between lytic growth and lysogeny. During lysogeny, it can integrate virtually anywhere in a chromosome. Once integrated, it can transpose itself to another location. (Ex. IT can jump fmo a phage genome to chromsome or plasmid)

The lysogenic phase genome is called what?

A prophage

Describe the mechanism of conjugation. Do both daughter cells harbor the R plasmid? Do both cells receive a copy of the plasmid?

A) Transfer of DNA occurs through a special form of replication, called TRANSFER REPLICATION: 1) Replication initiates at the origin of transfer (oriT) located on the R plasmid and proceeds by the rolling circle mechanism. 2) CONJUGATION bridge forms between donor and recipient cell (donor cell literally senses presence of recipient): contains proteins that "nick" one DNA strand, and then pull it away from its complement through the bridge 3) The strand left in the donor is replicated to reform the double-stranded R plasmid & The transferred strand recircularizes and replicates into a double-stranded R plasmid. 4) The cells now detach: both the donor and recipient cells possess a complete copy of the R plasmid. 5) Each can go on to mate with other R- recipients. B) YES: The R plasmid consists of circular double-stranded DNA that can replicate autonomously. Therefore, it can be passed onto both daughter cells during cell division. C) NO - The donor cell harbors an R plasmid; the recipient cell does not.

What is the simplest transposable elements?

An insertion sequence

What are the two lifestyle choices a bacteriophage has?

BACTERIOPHAGE LIFESTYLE CHOICES LYTIC/PRODUCTIVE Infection: phage infects host --> phage replicates itself --> lyses the host cell --> releases progeny phage LYSOGENIC Infection "temperate phage": phage becomes latent DOES NOT REPLICATE first--> circularize or integrate into the host chromosome --> latent genome called prophage --> in response to stressor can switch to lytic infection

What is another name for the bacterial virus genome? Is it made up of RNA or DNA? Is it double or single stranded? Linear or circular? What is its size range? What are its lifestyle cohices? What is its clinical relevance?

Bacteriophage, phage either RNA or DNA either double- or single-stranded either linear or circular 3 to 300 kilobases Lytic vs. lysogenic infection CLINICAL RELEVANCE: can carry virulence factors e.g., Diptheria Beta-toxin or Cholera toxin*

What is the function of a sex pillus? What is the function of a conjugation bridge?

Both are part of the mechanism of conjugation 1) Sex pillus: Facilitate capture of recipient cell to form a mating pair 2) Conjugation bridge: forms between donor and recipient cell, DNA passes through it from donor --> recipient

Gene exchange systems can move what? They permit transfer of what between what?

CAN MOVE TRANSPOSONS BETWEEN CELLS permits transfer of resistance --from cell-to-cell --within species --from one species to another

A conjugal transposon codes what type of genes? What can they transfer between In what microbial species is it found?

CONJUGAL TRANSPOSON --Encode both: A) genes for transfer machinery for conjugation between two IS elements B) Antibiotic resistant genes --Thus, a conjugal transposon from a normal human flora could be transferred to a pathogenic bacteria, conferring both the ability to transpose within a new host as well as transferring resistance by conjugation They can transfer within a given cell, or even mediate their own transfer to other cells! found in Streptococcal Enterococcus facaelis & many Bacteroides species

Between what are conjugative plasmids transfered? What is their clinical relevance? What is true of transfer of NON-conjugative plasmids? What is their clinical relevance?

CONJUGATIVE PLASMIDS: autonomous transfer from host to host within or between species --CLINICAL RELEVANCE: major cause of spread of multiple antibiotic resistances NON-CONJUGATIVE PLASMIDS: cannot transfer themselves, BUT can be transferred by conjugative plasmids --often used for genetic engineering

How could a transposon's genetic material move between cells? What is the clinical relevance of this?

Clinical Relevance of Transposition. Transposons are a major carrier of antibiotic resistance genes & have participated in the evolution of R plasmids. -- Since they can jump from one genome to another, they can find their way to phage and conjugal plasmids. --In this manner, they can participate in the general transfer of genetic material from cell to cell and from species to species.

What class of bacteria carry R plasmids? Can they carry only 1 or more resistances? What is so dangerous about selection for antibiotic resistance with R plasmids? Can they transfer between species? Why are nonpathogenic bacteria carrying plasmids so dangerous?

Conjugal R plasmids (also called RTF or Resistance Transfer Factor) many Gram-negative bacteria carry R plasmids that carry multiple resistances thus, selection for one antibiotic can cause selection for multiple antibiotic resistance can transfer between species nonpathogenic bacteria that carry R plasmid often act as reservoir to pathogenic bacteria

In order for bacterial AND eukaryotic cells, what must occur for transcription to be active?

DNA must be decondensed so that RNA polymerase can gain access.

What are the four major types of transposable elements?

Four major types of transposable elements exist: i) insertion sequence (IS) elements ii) transposons iii) transposable prophages iv) conjugal transposon

How could transposon move from host to host?

Gene exchange systems can move transposable elements between cells/hosts but transposable elements don't move themselves from one host to another (only within cells) Ex. A bacteriophage, once it becomes lytic, could enter another cell and then jump from the phage genome to a chromsome or plasmid.

What type of genes do the R plasmid encode that facilitates conjugation?

Genes carried by the R plasmid encode a SEX PILLUS (longer and thicker than the common pilus), which facilitates capture of the recipient cell to form a mating pair. A conjugation bridge forms between these two cells through which DNA passes.

Describe organization of the chromsomal genome in broad terms

It is bound by polyamines and specialized DNA-binding proteins that pack it tightly. It is folded: each fold is attached to some central structure and is independently supercoiled.

When antibiotics were fist introduced, why was it believed that abx resistance would not develop during therapy?

It was thought that frequency of mutation to resistance in bacteria was too low.

What was the traditional view of the bacterial chromosome? Is it right or wrong?

Loosely organized "tangled knot of DNA" often contrasted to the highly ordered structure of the typical eukaryotic chromosome WRONG! It is an artifact of the methods used to liberate the NUCLEOID, whose compact structure is not as stable as that of the eukaryotic chromosome. It took the development of gentle isolation protocols and the application of high resolution physical techniques such as Atomic Force Microscopy to show that the nucleoid is - instead - highly organized very much like the eukaryotic chromosome.

What is the clinical relevance of conjugation?

Many important antibiotic resistance determinants are carried by conjugal plasmids called R plasmids. 1) rapid dissemination of drug resistance via conjugal R plasmids or conjugal transposons (resistance can be to MULTIPLE abx: chloramphenicol, streptomycin, ampicillin, kanamycin and neomycin). 2) MEdiates intra-cell transfer AND can also mediate inter-species transfer from non-pathogenic Bacteroides to pathogenic Shigella or E. coli or Salmonella or worse 3) Normal flora acts as resistance reservoir* Thus, a single multiply resistant conjugal plasmid can disseminate rapidly.

Do all bacteria have a simple genome?

No - E. coli has a simple genome, but Borrelia (which causes Lyme disease) has 1 linear chromsome, 9 circular plasmids, and 13 linear plasmids Thus - bacteria have have linear OR circular chromsomes, and both can vary in number.

Why is the bacterial genome called a nucleoid?

Not surrounded by a membrane like a nucleus is

How do phages infect cells? Compare lytic vs. lysogenic infection

Phage Infection: 1) A phage infects a sensitive cell by first adsorbing (binding) to a specific receptor on that cell's surface and then by injecting its genome into the interior of the cell. 2) Two types of phage exist. They are named for the fate of the phage genome: 1) lytic or 2) temperate Lytic Infection phage replicates itself proteins synthsized and genome organized into a capsid lyses the host cell releases progeny phage which reiterate process Lysogenic Infection phage becomes latent does not replicate phage genome called prophage can circularize or integrate into the host chromosome cell that carries prophage is called a lysogen

What is the relationship between transposons and R plasmids?

R plasmids are built to a large extent by transposable elements that carry resistance genes Transposition manages to get them all to build together into one plasmid that can move from one host to another through a system of gene exchange called conjugation

How do R plasmids evolve to acquire resistances? How can they amplify themselves? An R plasmid that acquires what ability becomes particularly troublesome?

R plasmids evolve quickly, easily acquiring resistances by: -- fusing to other plasmids --acquiring transposons. They can amplify themselves by --duplication --or by increasing the number of plasmids. An R plasmid becomes particularly troublesome if it acquires the ability to mediate their own transfer from one cell to another through conjugation.

How does resistance conferred by R plasmids contrast from resistance conferred due to chromsomal mutations?

R plasmids often encode enzymes that i) inactivate antibiotics or ii) reduce permeability to antibiotics In contrast, resistances conferred by chromosomal mutation usually involves the modification of the antibiotic target, e.g. RNA polymerase and streptomycin

Why can't normal human viruses infect bacteria?

Simple membrane fusion wouldn't get the virus into the bacterial cell due to its peptidoglycan layer.

What is transduction mediated by and what this agent composed of?

TRANSDUCTION mediated by bacteriophage composed of: 1) protein capsid 2) DNA or RNA genome

What two things does transformation involve? What must recipient cells be? What is the clinical relevance of it? Can both gram + and gram - undergo transformation?

TRANSFORMATION 1) naked DNA released by donor cell (lysis) 2) uptake of DNA by recipient cell (competence) Recipient cell must be COMPETENT: able to take up DNA from environment CLINICAL RELEVANCE Streptococcus pneumoniae, Haemophilus influenzae & Neisseria gonorrhea use transformation to alter appearance of clinically important surface antigens --> permits evasion of host immune system* Mechanisms differ somewhat between Gram-positive and Gram-negative bacteria, but the principle is fundamentally the same.

Describe the clinical implications of gene expression with regard to antibiotic resistance

The genetic ecology of antibiotic resistance is complex. Bacteria co-existing in the lower GI tract, in the soil, and in other environments can readily exchange DNA encoding diverse antibiotic resistances. In addition, DNA is actually quite stable in the environment, especially when attached to particulate matter. Antibiotic preparations themselves are often contaminated with DNA that encodes resistance genes. This contamination thus increases the chance of genetic exchange via transformation between antibiotic-producing organisms and the pathogens they are being used to control

What is a prophage? What can it do?

The lysogenic phage genome Can circularize and remain autonomous (like plasmids) or integrate into the host chromsome

What are the 3 fates of the exogenate during transformation? Which possibility is most clinically relevant?

The remaining strand now proceeds through 1 of 3 fates i) degradation by nuclease (there will be no heritable change in the recipient) ii) circularization: lost by dilution (note, cannot replicate on its own. If cell replicates, it will be inherited as a plasmid by ONE daughter cell only - not both) iii) recombination into the chromosome (stably retained): donor DNA REPLACES recipient DNA --this possibility is most clinically relevant b/c if the recombinant gene contains antibiotic resistance or some other survival advantage, then the cell with recombinant DNA can be selected for!

How do bacterial cells acquire resistance that requires multiple changes? How did researchers dtm that single point mutations could not be causing antibiotic resistance to multiple abx at the same time?

They do so by acquiring large pieces of DNA from other bacteria or from bacterial viruses (also called bacteriophages) through genetic exchange: -transformatoin -conjugation -transduction To gain resistance to multiple antibiotics from single point mutations would require multiple single point mutations -- the probability of this happening was VERY small. Thus, another mechanism had to be leading to the fact that a bacteria could become resistant to multiple anitbiotics simultaneously

Transposons are transposable elements that carry genes for what? Are they smaller or larger than IS? What is the main mechanism of transfer?

Transposons are transposable elements containing genes necessary for transposition and for a variety of other functions including resistance to antimicrobial agents and heavy metals. They are considerably larger than IS elements, because they are hybrids that consist of a central core of genes bounded by two IS elements. Conjugation is the means of transfer. Conjugal transposons are found in organisms as diverse of Streptococcus and Bacteroides.

Are all genomes made of DNA? What is the exception?

Yes - all genomes are made of DNA with the exception of some viruses

What is a recombinase? What type of transposable element carries it?

a transposition enzyme (transposase) carried by insertion sequence

What do R plasmids carry? They oftne encode enzymes that do what 2 things? In what class of bacteria are they found??

carry multiple antibiotic resistance genes resistance genes often encode enzymes that i) inactivate antibiotics or ii) reduce permeability to antibiotics found in gram NEGATIVE bacteria

What is a bacteriophage composed of? What form of gene exchange does it mediate? What is its clinical relevance?

composed of: 1) protein capsid 2) DNA or RNA genome (can't be both) TRANSDUCTION is mediated by bacteriophage CLINICAL RELEVANCE Phage can carry virulence factors --Beta - toxin produced by Corynebacteria diptheria Beta - phage (toxin causes diptheria) OR --Cholera toxin produced by Vibrio cholerae CTX phage* To cause the toxic effects of diphteria and cholera, the host bacterium must be infected by a phage that contains genetic information for production of these toxins. Infected cells are virulent; those that are not are avirulent.

What is "exogenote" and "endogenote"

donor DNA = exogenote recipient DNA = endogenote

What is the relatinoship between a tranpson and an insertion sequence?

transposons are considerably larger than IS elements, because they are hybrids that consist of a central core of genes bounded by two IS elements transposition occurs by recognition by the recombinase of the terminal inverted repeats and a target area into which a new copy of the IS element will be inserted. If a transposon with two insertion sequences with two transposable genes are now made, by evolution, one of those transposase genes will mutate so it doesn't make a transposase any longer, and the 2 internal parts of the inverted repeats will evolve so they are no longer recognized by the remaining transposase. This leaves you with 1 transposase and only the inverted repeats on the outside, allowing this transposon of combined to IS to now act as a unit. Thus, you begin with 2 independent insertion sequences and end up with something that is ONE transposable element with one inverted repeat flanking it and one gene for a transposase.

How many bases does an insertion sequence element have? What type of genes do they carry? What do inverted repeats represent? Insertion into coding region of DNA (gene) causes what?

~ 1000 bases ONLY carry genes/sequences for --TRANSPOSITION, including its own site-specific recombinase (transposase) = transposition enzyme AND --2 INVERTED REPEATS (distinct nucleotide recognition sequences located at each terminus in inverted order) = recombinase recognition sites insertion into a gene causes insertion mutation