Siti Sarah Jumali 06-4832123 sarahjumali@ns.uitm.edu.my Genetic Transfer Siti Sarah Jumali 06-4832123 sarahjumali@ns.uitm.edu.my

Overview on Bacterial Gene Transfer Bacteria are usually haploid Makes it easy to identify loss-of-function mutations in bacteria than in eukaryotes These usual recessive mutations are not masked by dominant genes in haploid species Bacteria reproduce asexually Therefore crosses are not used in the genetic analysis of bacterial species Rather, researchers rely on a similar phenomenon called genetic transfer In this process, a segment of bacterial DNA is transferred from one bacterium to another

Genetic transfer A process to transfer genetic material from a bacterium to another bacterium Enhances genetic diversity Confer resistance to antibiotic when one a antibiotic resistant bacterium transfer the gene to another bacterial cell

Mechanism of Gene Transfer Conjugation Direct physical interaction between Donor and recipient cell Transduction When virus infects a bacterium and transfer genetic material Transformation Information is taken from a dead bacterium which releases it to the environment

Mechanism of Gene Transfer

CONJUGATION

CONJUGATION Direct physical interaction between Donor and recipient cell E.g plasmid is transferred to a recipient cell from a donor Requires the presence of a special plasmid called the F plasmid.

Conjugation cont’d A “mating” process between a donor F+ (bacteria with fertility factor =plasmid) and an F- recipient cell. Occurs in Gram - enteric bacteria like E.coli Plasmids carry genes that are nonessential for the life of bacteria. Uses pili (sex pilus). Eg. plasmid replication enzymes. Causes medical Problem: R-Factor = antibiotic resistance!

Conjugation Discovered in 1946 in bacteria by Joshua Lederberg and Edward Tatum They were studying strains of Escherichia coli that had different nutritional growth requirements Auxotrophs cannot synthesize a needed nutrient Prototrophs make all their nutrients from basic components One auxotroph strain was designated bio– met– phe+ thr+ It required one vitamin (biotin) and one amino acid (methionine) It could produce the amino acids phenylalanine and threonine The other strain was designated bio+ met+ phe– thr–

The genotype of the bacterial cells that grew on the plates has to be bio+ met+ phe+ thr+ Lederberg and Tatum reasoned that some genetic material was transferred between the two strains Either the bio– met– phe+ thr+ strain got the ability to synthesize biotin and methionine (bio+ met+) Or the bio+ met+ phe– thr– strain got the ability to synthesize phenylalanine and threonine (phe+ thr+) The results of this experiment cannot distinguish between the two possibilities

The need for physical contact Bernard Davis later showed that the bacterial strains must make physical contact for transfer to occur He used an apparatus known as U-tube It contains at the bottom a filter which has pores that were Large enough to allow the passage of the genetic material But small enough to prevent the passage of bacterial cells Davis placed the two strains in question on opposite sides of the filter Application of pressure or suction promoted the movement of liquid through the filter

Many species of bacteria can conjugate The term conjugation now refers to the transfer of DNA from one bacterium to another following direct cell-to cell contact Many species of bacteria can conjugate Only certain strains of a bacterium can act as donor cells Those strains contains a small circular piece of DNA termed the F factor (for Fertility factor) Strains containing the F factor are designated F+ Those lacking it are F– Plasmid is the general term used to describe extra-chromosomal DNA Plasmids, such as F factors, which are transmitted via conjugation are termed conjugative plasmids These plasmids carry genes required for conjugation

Some info on plasmid Small, circular pieces of DNA that are separate and replicate independently from the bacterial chromosome. Contains only a few genes that are usually not needed for growth and reproduction of the cell. But important in stressful situations F plasmid, facilitates conjugation Can give a bacterium new genes that may help for survival in changing environment. Some plasmids can integrate reversibly into the bacterial chromosome. An integrated plasmid is called an episome.

Plasmid There are several types of plasmids: a. Conjugative plasmids – genes for sex pili and conjugation b. Dissimulation plasmids – genes for enzymes that catabolize unusual organic molecules (Pseudomonas species – toluene, camphor, petroleum products) c. Plasmids carrying genes for toxins or bacteriocins d. Plasmids carrying genes for resistance (R) factors i. Consist of two sets of genes – RTF (resistance transfer factor) and specific resistance genes (r-determinant)

Mechanism of Conjugation The first step in conjugation is the contact between donor and recipient cells This is mediated by sex pili (or F pili) which are made only by F+ strains These pili act as attachment sites for the F– bacteria Once contact is made, the pili shorten Donor and recipient cell are drawn closer together A conjugation bridge is formed between the two cells The successful contact stimulates the donor cells to begin the transfer process

F’ factors can be transferred through conjugation The result of conjugation is that the recipient cell has acquired an F factor Thus, it is converted from an F– to an F+ cell The F+ cell remains unchanged In some cases, the F factor may carry genes that were once found on the bacterial chromosome These types of F factors are called F’ factors F’ factors can be transferred through conjugation This may introduce new genes into the recipient and thereby alter its genotype

Hfr Strains In the 1950s, Luca Cavalli-Sforza discovered a strain of E. coli that was very efficient at transferring chromosomal genes He designated this strain as Hfr (for High frequency of recombination) Hfr strains are derived from F+ strains

Mechanism in Hfr Strains

William Hayes demonstrated that conjugation between an Hfr and an F– strain involves the transfer of a portion of the Hfr bacterial chromosome The origin of transfer of the integrated F factor determines the starting point and direction of the transfer process The cut, or nicked site is the starting point that will enter the F– cell Then, a strand of bacterial DNA begins to enter in a linear manner

The F– cell does pick up chromosomal DNA It generally takes about 1.5-2 hours for the entire Hfr chromosome to be passed into the F– cell Most matings do not last that long Only a portion of the Hfr chromosome gets into the F– cell Since the nick is internal to the integrated F factor, only part of the plasmid is transferred and the F– cells does not become F+ The F– cell does pick up chromosomal DNA This DNA can recombine with the homologous region on the chromosome of the recipient cell This may provide the recipient cell with new combination of alleles

Hfr (High Frequency Recombination) Hfr- bacterial plasmid integrates into the chromosome. Medical Problem: Hfr antibiotic resistance genes are passed during binary fission (every time the cell divides). Therefore, antibiotic resistance spreads very rapidly! When Hfr mate with F – bacteria, only the bacterial genes cross NOT plasmid genes. Genetic diversity results in this case due to recombination.

Hfr (High Frequency Recombination)

Interrupted Mating Technique Developed by Elie Wollman and François Jacob in the 1950s The rationale behind this mapping strategy The time it takes genes to enter the recipient cell is directly related to their order along the bacterial chromosome The Hfr chromosome is transferred linearly to the F– recipient cell Therefore, interrupted mating at different times would lead to various lengths being transferred The order of genes along the chromosome can be deduced by determining the genes transferred during short matings vs. those transferred during long matings

Wollman and Jacob started the experiment with two E. coli strains The donor (Hfr) strain had the following genetic composition thr+ : Able to synthesize the essential amino acid threonine leu+ : Able to synthesize the essential amino acid leucine azis : Sensitive to killing by azide (a toxic chemical) tons : Sensitive to infection by T1 (a bacterial virus) lac+ : Able to metabolize lactose and use it for growth gal+ : Able to metabolize galactose and use it for growth strs : Sensitive to killing by streptomycin (an antibiotic) The recipient (F–) strain had the opposite genotype thr– leu– azir tonr lac – gal – strr r = resistant

Wollman and Jacob already knew that The thr+ and leu+ genes were transferred first, in that order Both were transferred within 5-10 minutes of mating Therefore their main goal was to determine the times at which genes azis, tons, lac+, and gal+ were transferred The transfer of the strs was not examined Streptomycin was used to kill the donor (Hfr) cell following conjugation The recipient (F– cell) is streptomycin resistant

From these data, Wollman and Jacob constructed the following genetic map: They also identified various Hfr strains in which the origin of transfer had been integrated at different places in the chromosome Comparison of the order of genes among these strains, demonstrated that the E. coli chromosome is circular

TRANSDUCTION

TRANSDUCTION The transfer of genetic material from donor bacteria to recipient bacteria via transducing agent (bacterial viruses called bacteriophage). Discovered in 1952 by Zinder & Lederberg. Two kinds of transduction: generalized and specialized.

Transduction A bacteriophage is a virus that specifically attacks bacterial cells It is composed of genetic material surrounded by a protein coat It can undergo two types of cycles Lytic Lysogenic

It will switch to the lytic cycle Prophage can exist in a dormant state for a long time Virulent phages only undergo a lytic cycle Temperate phages can follow both cycles

Transduction Phages that can transfer bacterial DNA include P22, which infects Salmonella typhimurium P1, which infects Escherichia coli Both are temperate phages

Generalized transduction Starts with the LYTIC CYCLE where a T- even phage infects E. coli killing the host cell, and synthesizing 2,000 copies of itself. The T-even phage randomly packages bacterial DNA in a few defective phages. Once a T –even phage infects another E. coli, this genetic information can be recombined into the host cell without causing the lytic cycle. New genetic information is thereby transduced from one bacteria to another.

Generalized Transduction

Generalized Transduction

Specialized Transduction Lambda phage infects E.coli but does not lyse the cell immediately. Instead it integrates into chromosome of the bacteria as a prophage and remains dormant. This is called the LYSOGENIC CYCLE. Phage genes are replicated and passed to all daughter cells until the bacteria is under environmental stress, from lack of nutrients, etc. Then phage gene will excise from the nucleoid and enter the LYTIC CYLE taking one adjacent gene for galactose metabolism.

Specialized Transduction cont’d The gal transducing phage (lambda) makes ~ 2,000 copies of itself with the gal gene, and infects other E.coli. When gal integrates into the nucleoid of other E. coli, it may provide these bacteria with a new capacity to metabolize galactose.

Specialized Transduction Graphic

Comparison of Bacteriophage Comparison of bacteriophage transduction in E.coli. Generalized Specialized T even phage lambda phage lytic cycle lysogenic random packaging specific gal gene

TRANSFORMATION

TRANSFORMATION The passage of homologous DNA from a dead donor cell to a living recipient cell. Occurs in Streptococcus pneumoniae. When S. pneumo dies the DNA can be absorbed by a living S. pneumo and recombined into the chromosome. The gene for capsule formation is obtained in this way, as is a gene for penicillin resistance. Discovered in 1929 by Fredrick Griffith.

Griffith’s Transformation Experiment

Griffith’s experiment (a) Inject living encapsulated bacteria into mice, mice die, encapsulated bacteria isolated from dead mice. (b) Inject living nonencapsulated bacteria into mice, mice remain healthy, a few non-encapsulated bacteria can be isolated from the living mice – most phagocytized by leukocytes. (c) Inject heat-killed encapsulated bacteria into mice, mice remain healthy, no bacteria isolated from the living mice. (d) Inject living non-encapsulated and heat-killed encapsulated bacteria into mice, mice die, isolated encapsulated bacteria from dead mice.

The Experiments of Avery, MacLeod and McCarty Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material They carried out their experiments in the 1940s At that time, it was known that DNA, RNA, proteins and carbohydrates are major constituents of living cells They prepared cell extracts from type IIIS cells containing each of these macromolecules Only the extract that contained purified DNA was able to convert type IIR into type IIIS

Hershey and Chase Experiment with Bacteriophage T2 In 1952, Alfred Hershey and Marsha Chase provided further evidence that DNA is the genetic material Inside the capsid They studied the bacteriophage T2 It is relatively simple since its composed of only two macromolecules DNA and protein Made up of protein

Life cycle of the T2 bacteriophage

The Hershey and Chase experiment can be summarized as follows: Used radioisotopes to distinguish DNA from proteins 32P labels DNA specifically 35S labels protein specifically Radioactively-labeled phages were used to infect nonradioactive Escherichia coli cells After allowing sufficient time for infection to proceed, the residual phage particles were sheared off the cells => Phage ghosts and E. coli cells were separated Radioactivity was monitored using a scintillation counter

Transformation The process by which a bacterium will take up extracellular DNA released by a dead bacterium It was discovered by Frederick Griffith in 1928 while working with strains of Streptococcus pneumoniae There are two types Natural transformation DNA uptake occurs without outside help Artificial transformation DNA uptake occurs with the help of special techniques

Transformation Graphic

TRANSPOSITION Transposons (jumping genes) are big chunks of DNA that randomly excise and relocate on the chromosome. Transposons were discovered in 1950 by Barbara McLintock in corn. Causes antibiotic resistance in Staph. aureus, the famous methicillin resistant Staphlococcus aureus (MRSA) strain!

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