Plasmid genetics

Plasmid genetics slide has basics of properties of plasmid, types, amplification, curing and plasmid incompatibility

1. Plasmid Genetics Vishrut S. Ghare ( M.Sc Microbiology, SET) Asst. Professor, S.B.B alias A. Jedhe College, Pune

2. Plasmid genetics i . Types of plasmids ii. Properties of Plasmid iii. Plasmid replication iv. Plasmid incompatibility v. Plasmid curing vi. Plasmid amplification Concept

3. Structure and Properties of plasmids In addition to having a chromosome, many bacteria possess plasmids are small, extra-chromosomal, often covalently closed circular DNA molecules. The existence of plasmids in bacterial cytoplasm was revealed by Joshua Lederberg in 1952 while working on conjugation process in bacteria. Lederberg coined the term ‘plasmid’ to refer to the transmissible genetic elements that were transferred from one bacterial cell to another and determined the maleness in bacteria. Some plasmids are present in many copies per cell, whereas others are present in only one or two copies. They are not essential for the bacterium but may confer a selective advantage during adverse environmental conditions. In general, plasmids carry genes that are not essential to bacterial function but that may play an important role in the life cycle and growth of their bacterial hosts.

4. Borrelia burgdorferi , the causative agent of Lyme disease in humans, has a 0.91-Mb (1 Mb = 1 megabase = 1 million base pairs) linear chromosome and at least 17 small plasmids, some linear and some circular, with a combined size of 0.53 Mb. Rhizobium radiobacter (formerly called Agrobacterium tumefaciens ), the causative agent of crown gall disease in some plants, has a 3.0-Mb circular chromosome and a 2.1-Mb linear chromosome. Among the archaea , chromosome organization also varies, although no linear chromosomes have yet been found. For example, Methanococcus jannaschii has a 1.66-Mb circular chromosome, and 58-kb and 16-kb circular plasmids, and Archaeoglobus fulgidus has a single 2.2-Mb circular chromosome.

5. There are many different types of plasmids; E. coli alone is estimated to have more than 270 different naturally occurring plasmids. Some plasmids promote mating between bacteria; others contain genes that kill other bacteria. Episomes : Sometimes F factor plasmid gets integrated into bacterial chromosome, such plasmids are called episomes . Such cells are Hfr , i.e. high frequency of recombination. Properties : Plasmid is an extra-chromosomal element, often a covalently closed, circular DNA. It exists in 3 different fashions – a. Closed circular b. Open circular c. Linear DNA Closed circular – if both the strands of DNA molecule are intact circles then molecule is described as covalently closed circular DNA or CCC DNA Open circular – If only one strand is intact then the molecule is open circles or OC DNA.

6. Typical Plasmid has 3 important elements - a. An origin of replication b. A Selectable marker gene (Antibiotic resistance) c. A cloning site (a place to insert foreign DNA)

7. Naturally occurring plasmids vary in size from approximately 1 kilobase to more than 1 megabase . Replicons - They replicate independently of the bacterial chromosome. Replication of plasmid takes place via rolling circle mechanism . Plasmids can transferred from one bacterium to another ( may be of another species) via 3 different mechanisms of horizontal gene transfer: transformation, transduction and conjugation. Unlike viruses (which encase their genetic material in capsid ) plasmids are naked DNA don't encode gene necessary to encase the genetic material for transfer to a new host. However some classes of plasmids encode the conjugative sex pilus necessary for their own transfer. Size; ranges from; less than 1x10 6 Daltons to greater than 200x 10 6 Daltons Plasmids confer phenotypic traits on their host cells. Certain plasmids have not yet been ascribed any phenotypic traits, called cryptic plasmids.

8. Plasmids can be conjugative and non-conjugative Conjugative: carry transfer gene ( tra gene), promote bacterial conjugation, also known as self transferable ex= R and F plasmid Non-conjugative: do not carry tra gene, These plasmids are know as Non-self-transferable ex= ColE1 and pSC101 Generally conjugative plasmids have high molecular weight and are present as 1 to 3 copies per chromosomes Non-conjugative: low mol weight and multiple copy Exception: conjugative plasmid R6K, mol wt is 25x 10 6 Daltons and maintained as relaxed plasmids Relaxed plasmids: Multiple copies of plasmids per cells are referred as relaxed Stringent plasmids : Limited number of copies of plasmids per cells are referred as stringent

9. Types of plasmids 1. F-plasmid (F-factor): F-plasmid or F-factor (“F” stands for fertility) is the very well characterised plasmid. It plays a major role in conjugation in bacteria E. coli and was the first to be described. It is this plasmid that confers ‘maleness’ on the bacterial cells; the term ‘sex-factor’ is also used to refer to F-plasmid because of its this property. F-plasmid is a circular dsDNA molecule of 99,159 base pairs. One region of the plasmid contains genes involved in regulation of the DNA replication (rep genes), the other region contains transposable elements (IS3, Tn 1000, IS3 and IS2 genes) involved in its ability to function as an episome , and the third large region, the tra region, consists of tra genes and possesses ability to promote transfer of plasmids during conjugation. Example F-plasmid of E. Coli.

10. 2. R-plasmids: RTF –resistance transfer factor R-plasmids are the most widespread and well-studied group of plasmids conferring resistance (hence called resistant plasmids) to antibiotics and various other growth inhibitors. R- plasmids typically have genes that code for enzymes able to destroy and modify antibiotics. Some R-plasmids possess only a single resistant gene whereas others can have as many as eight. Plasmid R 100, for example, is a 94.3 kilobase -pair plasmid that carries resistant genes for sulfonamides, streptomycin and spectinomycin , chloramphenicol , tetracyclin etc. It also carries genes conferring resistance to mercury. Many R-plasmids are conjugative and possess drug- resistant genes as transposable elements, they play an important role in medical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections.

11. Some strains showed multiple resistant to six drugs as chloramphenicol (Cm), tetracycline ( Tet ), streptomycin ( Sm ), sulfisomidine (Su), ampicillin (Amp), and trimethoprim ( Tp ) Resistance.

12. 3. Virulence-plasmids: Virulence-plasmids confer pathogenesity on the host bacterium. They make the bacterium more pathogenic as the bacterium is better able to resist host defence or to produce toxins. For example, Ti-plasmids of Agrobacterium tumefac iens induce crown gall disease of angiospermic plants; Entertoxigenic strains of E. coli cause traveller’s diarrhoea because of a plasmid that codes for an enterotoxin which induces extensive secretion of water and salts into the bowel. Ex: K88 plasmid of E. coli

13. 5. Metabolic or Degradative plasmids: Metabolic plasmids (also called degradative plasmids) possess genes to code enzymes that degrade unusual substances such as toluene (aromatic compounds), pesticides (2, 4-dichloro- phenoxyacetic acid), and sugars (lactose). CAM and TOL plasmid of Pseudomonas putida is an example which metabolizes camphor and toulene respectively. However, some metabolic plasmids occurring in certain strains of Rhizobium induce nodule formation in legumes and carry out fixation of atmospheric nitrogen. 6. Col plasmids: Col plasmid in E. Coli encodes for colicins a type of bacteriocin . They carry gene which codes for bacteriocin (bacterial proteins that destroys bacterial cells) Ex: Col E1 of E. Coli and Col E2 of Shigella sp .

14. Plasmid incompatibility Plasmid incompatibility  refers to the inability of two  plasmids  to coexist stably over a number of generations in the same bacterial cell line. Generally, closely related  plasmids  tend to be  incompatible , while distantly related  plasmids  tend to be compatible. Plasmid incompatibility is usually defined as the failure of two co-resident plasmids to be stably inherited together in the absence of external selection.  In simpler terms, if the introduction of a second plasmid negatively effects the inheritance of the first, the two are considered to be incompatible. Generally, two closely related plasmids cannot coexist in a bacterial cell. In the population of progeny cells derived from a cell containing two such plasmids, the proportion of cells having only one of the two plasmids increases with every cell division. This is known as plasmid incompatibility.

15. On the other hand, two different unrelated plasmids, e.g. F plasmid and ColEl can exist together without any difficulty, because these plasmids belong to two different incompatibility groups. Whereas, two F-plasmids cannot coexist in the same cell.

16. Plasmid Curing Definition - The elimination or loss of a plasmid from a cell culture occurs naturally through cell division or by treating the cells with any chemical or physical agents. Various methods involving chemical and physical agents have been developed to eliminate plasmids. Protocols for curing plasmids consist frequently of exposure of a culture to sub-inhibitory concentrations of some chemical agents, e.g. acridine orange, acriflavine , and sodium dodecyl sulfate or to a super-optimal temperature followed by selection of cured derivatives. In the instances where the plasmid is stable or the loss of property difficult to determine, the bacteria can be treated with curing agents. These include chemical and physical agents, some of which can mutate DNA, interfere specifically with its replication, or affect particular structural components or enzymes of the bacterial cell.

17. The elimination of a plasmid (curing) from a bacterial culture is the best method to substantiate the relationship between a genetic trait and carriage of specific plasmid by the culture as the phenotypic characters which are associated with the plasmid are not expressed in cured derivatives but on the re-introduction of the plasmid in to the cured strain the lost phenotype is re appeared. The efficiency of curing can also vary widely depending on the plasmid and the particular bacterial host carrying it. In most instances, the underlying mechanism of curing is not known. The agent may interfere directly with plasmid replication as occurs with the heat induced curing of certain temperature sensitive palsmid or curing of them by acridines or ethidium bromide. Alternatively, curing may interfere with the growth of plasmid carrying bacteria thereby allowing spontaneously arising plasmidless seggregants to become predominant. This occurs in certain instances of curing by acridines , sodium dodecyl sulphate and urea. Curing by Mitomycin C is also effective.

18. Plasmid curing procedure : 1. Some plasmids undergo spontaneous segregation and deletion. However, the majority are extremely stable, and require the use of curing agents or other procedures (elevated growth temperature, thymine starvation), to increase the frequency of spontaneous segregation. 2. Overnight, logarithmically growing culture is used to inoculate a series of culture flasks or test tubes containing nutrient broth or appropriate growth medium amended with a range of curing agent concentrations. 3. Cultures are incubated overnight (or longer if necessary) at the optimum or otherwise appropriate growth temperature for the particular strain under investigation. 4. Culture tube or flask displaying observable turbidity and containing the highest concentration of curing agent is used to plate out dilutions on nutrient agar or other similar medium.

19. 5. Individual colonies can be replica plated or patch-plated to a selective agar medium to screen for loss of plasmid-encoded trait (e.g., antibiotic or metal sensitivity) and/or individual colonies can be tested for plasmid loss using a micro scale plasmid isolation procedure. 6. Intercalating dyes, Coumermycin and novobiocin , Rifampicin , Mitomycin C, Sodium dodecyl sulfate , Elevated growth temperature are the plasmid curing agents used.

20. Plasmid Amplification: Another important point of plasmid replication is that chromosomal DNA synthesis and plasmid DNA synthesis are independent of each other, though, in both, DNA synthesis is followed by replication. Thus it is possible to stop chromosomal DNA synthesis and replication without affecting plasmid DNA synthesis and replication. Such situation can be practically created by adding chloramphenicol to a bacterial culture. This antibiotic specifically inhibits prokaryotic protein synthesis. When it is added to a growing bacterial culture, chromosomal DNA synthesis is inhibited, but plasmid DNA synthesis and replication continue at the cost of the available replication proteins which are not used for chromosomal DNA synthesis. The net result is that each bacterial cell contains large number of plasmid copies. This is known as plasmid amplification . When a specific gene which has been transferred (cloned) to a plasmid requires to be isolated, plasmid amplification becomes a useful tool, because of high plasmid DNA concentration in the total cellular DNA.