بسم هللا الرحمن الرحيم` Gene transferring in bacteria

Transcription

1 بسم هللا الرحمن الرحيم` Gene transferring in bacteria Bacterial genes (whether the bacteria are inside or outside our body) are exposed to changes. All types of commensal pathogenic bacteria are in steady genetic change, & according to the environment bacteria may exhibit transfer of genetic materials. For Example: We have a large number of bacteria in our intestines (a closed space), where the physical and chemical conditions may cause transfer of genes in particular bacterial species. The change of genes cannot be accomplished easily... There are certain factors that influence transfer of genes: 1- Developing of resistant bacteria: because it may convert a susceptible (vulnerable) kind of bacteria to a resistant one or a multi resistant one. 2- Any factors acquired during transfer of genes Example: Making the bacteria attach more firmly to mucosa by acquiring genes responsible for making specific enzyme or toxin. Mechanisms of transferring of genes in bacteria: 1- Transformation: - In bacterial life we always find cells that replicate and others that die or get eliminated. - The cells that die will leave segments of DNA and segments of plasmid. These segments are found in association with a few number of genes, and will be integrated into a living bacterial cell chromosome in homological recombination conditions. The conditions of recombination and integration are: 1-The DNA fragments carry specific insertion sequences. 2-The sequences must be homologous. Under these conditions integrating and accepting will occur. 1 Dr Asem

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3 -The transformation process is carried out by special types of bacteria that have special enzymes in their cytoplasm These special enzymes are mainly restriction enzymes which have two types: 1-Endonucleases which produce cuts within the bacterial chromosome. 2-Recombinases which allow these new segments to be integrated. This process also depends on the environment and where the segments are fragmented. Note: The released linear DNA can t survive in the environment and must enter a cytoplasm and attach to a chromosome. If the segment didn t manage to be integrated it will be eliminated from the bacterial cytoplasm. Any bacterial cell can t accept a lot of foreign DNA; it can only take 1-2% of its total DNA. Transformation is more important in gram +ve bacteria than gram -ve bacteria, because the cell wall of the gram +ve bacteria has multiple layers of peptidoglycans and many specific protein binding elements which might help transfer the DNA material into the bacterial cell. Examples: - An example of gram +ve bacteria that lives in our bodies and carries out this process (transformation) is streptococcus bacteria which is commonly present in our respiratory tract and usually not considered pathogenic. However, in certain conditions, it may be activated and become a source of infection and cause Pneumonia. It is often acquired from small segments of DNA in the surrounding medium. It can also change from capsulated form to non-capsulated form. - The same can also be applied to Staphylococcus, which can also be found in our noses and many parts of our bodies. It also acquires foreign segments of DNA. - We have a type of gram ve bacteria called Neisseria Gonorrhoeae, which easily acquires DNA segments and carries out the transformation process. -Finally, it is easy to find this process in the body, where we have many types of bacteria together. 3 Dr Asem

4 Examples: the oral cavity, respiratory tract, the intestines, and of course outside in vitro. B. Conjugation : which is associated with production of specific pilus that allow the transition of DNA segments or plasmid. Keep in mind that we have at least two types of plasmid: 1- Conjugative plasmid. 4 Dr Asem

5 2- Non-conjugative plasmid. A conjugative plasmid carries specific genes responsible for the process of conjugation. However, these plasmids might not manage to produce conjugation channels in gram +ve bacteria; it might only help to transfer segments of DNA from one bacterial cell to another. - This plasmid might be separated to small segments associated with specific regions and allow them to be attached to wall of bacteria and later enter the cytoplasm. - Because these segments carry a specific gene responsible for independent replication, and this independent replication must be associated with specific genes in order to survive, these small segments carry a specific operon as well as the gene responsible for circulation in order to produce a double stranded DNA. - This DNA later allows the segments to be present as extra-chromosomal DNA, therefore as you see this segment will be first produced as linear segment of DNA, but later, once it reaches the cytoplasm it will be slowly changed into circular form in order to survive inside the cytoplasm. [Please refer to picture] Inside the cell there's a specific linear plasmid, which can be transformed to survive in the cytoplasm and produce a STABLE plasmid in the cytoplasm. There are two ways for this to happen, either producing a plasmid or a small segmental DNA. In relation to conjugation, the process is more difficult and complicated, why? Because it s not easy to bring cells together (which is a huge step in the conjugation process) - This process take place in certain staph bacteria and gram ve Bacteria. Note: *Keep in mind, conjugation between Gram +ve and Gram ve is not possible. For a cell to conjugate with another, there must be a certain specific gene of conjugation, in relation to (depending on) the types of the bacteria (the type of the topology of the bacterial chromosome), which allows the cell to accept foreign DNA and accept the formation of pili. *The foreign DNA that is transferred to the cell can have, for example, a resistant gene which the cell can obtain in the process of conjugation. -Whether in vitro or in vivo, we must first have cells with a positive F-factor as a donor (which carries the gene responsible for the production of sex pili). Second, we must have a cell with a negative F factor to accept foreign DNA. -The process of conjugation, as you see in the picture, is 3 or 4 steps. 5 Dr Asem

6 On the left, there is a cell with a double stranded circular DNA, which we called a plasmid. This plasmid is carrying an F-factor. Usually F factors transcribe a gene to produce sex pilli. These pilli connect the two bacterial cells together, bringing the two cells together. Later, an enzyme opens the double stranded DNA (making it linear), creates a copy of the plasmid, and transfers it to the other bacterial cell. Due to the fact that this small segment carries the circulation enzyme (which is responsible for producing double stranded circular DNA in the cytoplasm) the single stranded linear transferred segment will become circular and double stranded, and the original copy, which also became linear and single stranded will again form a circular double stranded DNA. This process of conjugation has been recognized mainly in certain carriers, either in our body or outside our body: Inside the body bacteria should find certain conditions in order to survive. الصرف ( sewage Outside the body; usually it is found in areas related to polluted water in the where we find gram +ve and -ve bacteria. These bacterial cells are often ready to,(الصحي accept pilus, in order to complete the process of conjugation. * Generally under normal conditions, if we have a population of any bacteria in our intestine, the percentage of conjugation will not be more than 5 10% among all bacterial cells. Example: if we have 1 billion cells then the number of cells that will participate in conjugation process are million cells, but this is usually enough. * What is the significance of conjugation and transformation especially in the clinical microbiology?? - The most important point is the transfer of resistant factors against certain specific type of antimicrobes. - Note : transformation and conjugation might be associated with transfer of a certain specific gene, which is responsible for developing resistance. - when do conjugation and transformation cause a problem? When we give a course of treatment to a patient, it is widely known that an antibiotic course last for 2 days 4 weeks is expected to cure the patient from the bacteria. However, the patient might be cured from a part of bacteria that decays, but there are some bacterial cells that can resist by transformation and conjugation. 6 Dr Asem

7 So the major importance of the transformation and conjugation is to produce bacteria resistant to various conditions. (mechanisms of transfer) C. Transduction: which is the process of adding extra chromosomal DNA to the bacteria, using a specific virus, and this virus usually carries its own DNA in its head. This virus is called bacteriophage, which is not easily recognized as a vehicle for DNA transfer. Note: Bacteriophages are also considered as chemical viruses. The structure of the bacteriophage: 1. Head of the bacteriophage: consists of the capsid which contains the nucleic acid (either DNA or RNA) 2. Tail: looks like a syringe,(حقنة) associated with the tail fibers providing attachment with the receptors on the bacteria cell wall. Note : there is a specifity between bacteriophages and bacteria type. Note : attachment between bacteriophage and bacteria is specific ; ex:e.coli they have their own bacteriophage; so the E.coli bacteriophage cannot bind into cell wall of another type bacteria because it needs a specific receptor to attach. 7 Dr Asem

8 Once the bacteriophage attaches to the cell wall of its bacteria it injects small segment of its DNA ( NOT necessary all DNA ) because cannot accept large amount of foreign DNA. -Stages in brief: 1.attachment 2.injection of the foreign DNA 3. increase the number of bacteriophage inside the cytopalzim of the infected bacteria cell. regarding increase in number we have two mechanism : 1lytic/virulent phage 2-Lysogenic / Temperate phage Firstly ; lytic infection : once bacteriophage mange to introduce his DNA to the cytoplasm in linear form which is associated with number of genes that encode enzymes; these enzymes control all metabolic activity inside bacteria cytoplasm ( developing, production of aminoacids and polypeptides, structure etc ) Then within short period bacteria cells become controlled by gene inserted by bacteriophage and begin produce essential components of bacteriophage to increase in number which means that bacteria cell switch from produce its structural components to produce components of bacteriophage and after short time the cell will be filled with millions of bacteriophage which later will produce lytic enzymes that causing damage to the bacteria cell wall then these million will release out of the cell. -This lytic process in fact is important in nature especially because it contributes for decrease in number of contamination in water. -It is called natural purification of the water because as we know in the sewage we have large number bacteria, and these by the presence of bacteriophage and activity it will reduce the contamination with the bacteria in at least 10% or 20% or 30%. So the presence of bacteriophage is important to get rid of pollution and contamination specially in fluid medium. In the intestinal tract it is not so significant but still might be associated with transfer of resistant factors because during the lytic process some of these bacteriophages may capture gene cascade responsible for developing of resistant and this will be carried later again in the head of the bacteriophage and in the next generation it will associated with transfer of resistant factors. which means the bacteriophage may gain resistant against antimicrobial drugs in the intestinal tract of humans. 8 Dr Asem

9 2- Lysogenic / Temperate phage : it is more dangerous because the end product will incorporate small segment of DNA within the bacterial chromosome. And according to the type of the bacteriophage and the type of DNA in the head of the bacteriophage might certain specific genes introduce in the bacterial chromosomes and these might at the end converted bacteria to produce new product. So, lysogenicity is highly important in pathogenic bacteria. - Genes that are present within the DNA of the bacteriophage can be added to the chromosome of the bacteria, and translated by the bacterial cell into certain products. The importance of lysogenicity here is its ability to convert non-toxic bacterial cells into toxic ones. Example: we have at least two types of important bacteria in our respiratory and intestinal tracts that are non-toxic but pathogenic; such as Beta Hemolytic Streptococci and Streptococcus Pyogenes (group A streptococci). However, in children up to 12 years of age, who lack a well-developed immune system, these types of bacteria are highly pathogenic. They cause repeated sore throat (inflammation of tonsils) and tonsillitis, accumulating a certain antigenic structure of these bacteria. Therefore, this causes, at the end, what is called rheumatic fever which affects the heart. In addition, these strains of bacteria (group A streptococci) may become transformed into toxic bacterial cells. This happens after acquiring specific bacteriophages carrying certain genomes responsible for the production of toxins called erythrogenic toxins. During tonsillitis, children may suffer from skin infection caused by these toxins. The same symptoms apply to another type of bacteria called Corynebacterium which is the causative agent of Diphtheria. Before the era of triple vaccine (a vaccine against many infectious diseases including diphtheria), Corynebacterium was a major causative of childhood diseases, associated with killing of millions of children, since it released erythrogenic toxins, which cause severe damage in the heart muscle leading to myocarditis as well as other complications related to the respiratory tract. Corynebacterium, as a gram positive bacteria, is found in two forms: 1- Toxic form: which is the lysogenic form carrying a specific bacteriophage (which has genes responsible for the production of the toxins of the bacteria). 2- Nontoxic form: which is the major form of Corynebacterium. However, if the toxic form appears in the non-toxic population, it will replicate and spread in large numbers. 9 Dr Asem

10 Another example: Clostridium difficile, which is a spore forming bacteria. This bacteria can be in toxic and nontoxic forms. The lysogenic phage: It is also called temperate phage, because during the process of bacteriophage genome incorporation with the chromosomes of the bacterial cell, these genomes become a prophage for a short period of time, appearing in red color. This means it is not a mature phage. So, the prophage is the small segment in the bacterial chromosome that comes from bacteriophage. This segment might be separated during the replication, and once it s separated and released from dead bacterial cells, it might again produce a mature phage (transforming from a prophage into a mature phage). How? Because this phage carries genes responsible for production of all parts of the bacterial bactrophage the head, the tail, etc. The phage is actually a silent conform of DNA, containing genes within the bacterial chromosomes related to the lysogenic phage. In the end, it might be released. Now, how can we detect these bactrophages? Viruses as we know can now be detected by Microscope Simple culture media We have to use special devices in the laboratory. We have to get contaminated fluids from the sewage or a river, and we can investigate this water for the presence of bactrophage, by a method know as plaque essay: First we use culture media to increase these particles, which we cannot recognize in our eyes (the bacteria), in number. Then we do centrifugation and filtration. Finally, we have special plates with holes in them, in each hole we put a drop of this purified solution and we incubate it in 37 degree temperature. On the next day we detect the presence of clumps or colonies which are associated with bacteriophages, and we detect the type of bacteria. 10 Dr Asem

11 Example For E.coli, the plates are indicated with the presence of bactriophage and we can repeat this again in order to recognize the other types. -Features of Transduction: Transduction is important in clinical medicine in relation to developing of pathogenic and toxigenic types of bacteria.. Transduction is widely used in genetic engineering; they use the bacteriophage to introduce foreign human or animals DNA into a specific tissue. It is also used in treatment of malignancy. Production of specific materials like insulin. It is also highly important and widely used in molecular technique. Used to study types of diseases in relation to our human body It s important not only in developing resistance to diseases, but can also be used as a diagnostic method in clinical medicine. So, transduction is important in industry and important for bioengineering. Without transduction we would have not accomplished progress in clinical medicine. Uses of Transformation: Transformation is widely used in bioengineering specially to introduce specific genes in certain types of animals or plants Example: to change the color of a plant or change the end product of an organism or to introduce resistance to certain insecticides. In short: gene transfer in bacteria is not only related to clinical microbiology or to diseases, but in general bioengineering in our life. By using gene transfer and gene transduction we have accomplished a huge progress in studying the sequence of genes in different species including our human genome. How? Because the bacteriophage and conjugation can be used to sequence the genes in animal tissue and human tissue in types of living cells and this can help study different types of infection or types of diseases related to changing genes. The mechanism of gene transfer is highly important in medicine and we might in future treat many kinds of diseases only by manipulation of genes in our body. 11 Dr Asem

12 Done by : Mohammad Al-Khoujah Special Thanks For : Amjad Alsharabati, Owen Madaeen, Abdallah santarisi, Ali Altrabsheh, Ody Al Halhooli, Adel Muhannad and Laith Ejeilat. من يعيش االمل ال يعرف المستحيل 12 Dr Asem