bacterial plasmid, their propertis, classification

This presentation deals with the variety of plasmids, their general characteristics & their application in RDT

1. Devlina Sengupta Department of Microbiology Kanchrapara College Plasmids

2. Bacterial Plasmids The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952. A plasmid is a short, usually circular, and double- stranded segment of DNA that is found in the cytoplasm separate from the main bacterial chromosome.

3. The bacterial chromosome and bacterial plasmids, as shown in the electron microscope. The plasmids(arrow) are the circular structures, much smaller than the main chromosomal DNA.

4. Plasmid sizes vary from 1 to over 1,000 kbp. They usually contain 5 to 100 genes and usually carry genes that are useful but not essential to survival: e.g. genes which make bacteria resistant to antibiotics. As long as the bacterium is thriving in a low-stress environment, removing all the plasmids would not affect the ability of the bacterium to survive. Specifically, plasmids are nonessential, extrachromosomal pieces of DNA. Plasmid features

5. Classification Of Plasmids. I.Based on their ability to transfer to other bacteria. a) Conjugative plasmids - contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium. e.g., F plasmid, many R plasmid & some Col plasmid.

6. Non-conjugative plasmids - incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids. e. g., many R plasmid & most Col plsmid. Mobilisable plasmid - An intermediate class of plasmid. They carry only a subset of the genes required for transfer. They can parasitize another plasmid, transferring at high frequency in the presence of a conjugative plasmid.

7. Process of plasmid transfer The process by which non-conjugative plasmid is effectively transferred into a recipient via the contact provided by a conjugative plasmid is called donation . The process by which self transmissible plasmid via recombination carries along with it the non- mobilizable plasmid to a recipient is called conduction. Plasmid mobilization begins when a plasmid encoded protein, which is a nicking protein of the relaxation complex, makes a single strand break in a unique base sequence called the transfer origin or oriT . This nicking protein is attached at 5 ΄ end. This nick initiates rolling circle replication & the linear branch of the rolling circle is transferred. DNA synthesis occurs both in donor (called donor conjugal DNA synthesis that serves to replace the single strand that is transferred) & in recipient cells (called recipient conjugal DNA synthesis that converts transferred single strand to double stranded DNA)

8. tra genes of F Transfer of R plasmids immediately after receiving into a recipient cell is inhibited at many times due to a multisubunit repressor encoded by two fin genes which acts on an operator & prevents transcription of the genes required for transfer ( tra genes). This phenomenon is called fertility inhibition. Transfer of F plasmids is constitutive because they have repressor sequence disrupted by insertion of an IS3 sequence in finO . Thus R plasmid transfer is initiated into a recipient from a recent recipient, even before the repressor protein synthesis has started inside it. Most of the tra genes are controlled in an operon which is involved in pili synthesis.

9. II.Based on function. a) Degradative plasmids – They are able to digest unusual substances like toluene and salicylic acid. e.g.,TOL plasmid of Psedomonas putida. Virulence plasmids – contains vir genes which turn the bacterium into a pathogen. e. g., Ti & Ri plasmids Fertility (F)-plasmids - contain tra genes. They are capable of conjugation and result in the expression of sex pilli. Example: F plasmid of E. coli.

10. Resistance (R)plasmids – contain genes that provide resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood. e. g., pRP4 of Pseudomonas sp. Col plasmids - contain genes that code for bacteriocins & toxins that can kill other bacteria. e. g., ColE1

11. III. Based on copy number. a) Stringent Plasmid – It replicates only along with the main bacterial chromosome & is present as a single copy, or at most several copies, per cell. a) Relaxed Plasmid – It replicates within a cell independently of the chromosomal DNA replication. Thus multiple copies of plasmids are present.

12. Plasmid copy number The average number of a particular plasmid maintained inside a cell or the number of copies of plasmids in a newborn cell immediately after cell division. All plasmids must regulate their copy number, otherwise they would fill up the cell & become too great a burden for the host or their replication would not keep up with the cell replication & they would progressively be lost during cell division Plasmids with high copy number have different mechanism in copy number regulation as compared to plasmids with low copy number.

13. Control of plasmid copy number Regulation of copy number is by a repressor protein encoded by the plasmid itself, that negatively regulates the initiation of replication. Activity of this repressor is dependent on its own concentration. As the cell grows, volume increases so the repressor concentration drops & replication is not inhibited. Plasmid DNA molecules doubles. At this point the initial number of repressor genes doubles as well, causing larger amount of repressor protein synthesis & replication to stop. For high copy number plasmids complete repression requires a higher concentration of repressor than is required for low copy number plasmids. Each plasmid controls its own copy number. Some high copy number exhibit a phenomenon called Plasmid amplification whereby bacterial chromosomal DNA synthesis is inhibited by presence of a replication protein synthesis inhibitor & plasmid replication still continues in the absence of chromosomal DNA replication protein .

14. IV.Based on compatibility It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli . Eg : Plasmid ColE1 & plasmid F can coexist together because they fall under compatibility group/different incompatibility group i.e. repressor of one plasmid does not impact the replication of other plasmid. However , related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Thus, plasmids can be assigned into incompatibility groups based on the fact that repressor protein of one plasmid influences the replication of other plasmid & either of the plasmid is selected randomly for replication . In such cases as a single cell containing two compatible plasmids divides, the percentage of the progeny population containing only plasmid type will increase with each generation.

15. Plasmid replication Plasmids rely on host replication proteins for their replication. Some plasmids use host DNA pol III(like F) or some use host DNA pol I(like COLE1). Some replicate unidirectionally some bidirectionally (theta mode of replication). Plasmids replicating bi-directionally terminate replication in either of two ways: a) termination occurs when growing replication forks collide, b) termination occurs when one of the two replication fork reaches a fixed termination site. Plasmids have a separate mode of replication called “ Butterfly or rabbit’s ear mode ” whereby replicated molecule contains untwisted replicated portions & a supercoiled unreplicated portion. When replication cycle is completed, one of the circles is cleaved to separate the daughters in which newly replicated molecule is sealed & supercoiled later.

16. In image a) unidirectional replication is shown where origin region is designated by oriV . Replication terminates when the replication fork gets back to origin. In image b) bidirectional replication is shown where replication terminates when the replication forks meet at a point opposite oriV

17. Rolling circle replication where a nick is made at the double stranded origin (DSO) by the plasmid encoded Rep protein which remain bound to the 5’p at the nick. The free 3’OH serves as primer for the DNA pol III which replicates around the circle displacing the old strand as a ssDNA . The Rep protein makes another nick, releasing the single stranded circle. DNA ligase then joins the end to form ds & SS circular DNAs. The host RNA polymerase then makes a primer on SSDNA origin & then DNA pol III replicates SSDNA to make another ds circle. DNA Pol I removes the primer, replacing it with DNA & ligase joins the end to make ds circular DNA.

18. Plasmid curing Plasmid curing is the loss of plasmid during cell division. In general a plasmid with copy number n, the frequency of curing is 2(1/2) 2n considered when the sorting of the plasmid is completely random. ( 2 is multiplied because at the time of cell division the number of plasmid is twice the copy number). Several mechanisms exists to prevent plasmid curing like plasmid addiction system , site specific recombinases that resolve multimers or partitioning systems .

19. Plasmid Addiction

20. Plasmid partitioning Partitioning system ensures that a plasmid is not lost during cell division & atleast one copy of plasmid is segregated in each daughter cell. This is maintained by par function system of the cell. It consists of a cis acting site called ParS & two trans acting genes that encode proteins called ParA & ParB . ParS site is the site at which two copies of the plasmid are pulled apart during partitioning & parA & parB proteins act at this site. Although the mechanism of this action is still under research, but it is known that many copies of parB binds at around parS site blocking or silencing genes around this site. The parA protein next binds this complex of parBparS hydrolyzing ATP to ADP.

21. Two models for par site functions: Model a) plasmid binds to the unique site on the bacterial membrane & two copies of the plasmids are pulled apart as the site on the membrane divides. In model b) two copies of the plasmid bind to each other before they bind to a site on membrane. One then associates with each site when the site of the membrane divides.

22. Plasmid host range The host range of a plasmid includes all types of bacteria in which the plasmid can replicate & the host range is usually determined by the ori region. Some plasmids such as those with ori regions of the colE1 plasmid type including PBR322, pUC , pET are narrow host range plasmids as they replicate only in E.coli & some other closely related bacteria like Salmonella & Klebsiella species. Plasmids with broad host range includes RK2 & RSF1010 which will replicate in most gram negative bacteria & even in some gram positive bacterias . Some gram positive plasmids like pUB110 are also broad range plasmids as they would replicate in most gram positive bacteria.

23. F plasmids F plasmids apart from providing mobilizable function also acts in “ integrative suppression ” F plasmids have the ability to integrate at random sites(unlike λ phage’s site specific integration) into bacterial chromosome to generate an Hfr cell. F plasmids utilizes many proteins of host cells to replicate itself & excludes some other proteins involved in host chromosomal replication. One such protein is dnaA Bacterial chromosome mutant in replication protein called dnaA cannot replicate its own chromosomal DNA at high temperature (42 ̊C) but F plasmid with the help of other host replicative protein can continuously replicate itself at this temperature. In Hfr cells, however mutated dnaA protein called dnaA ( Ts ) is compensated by F integration as bacterial chromosome is able to replicate using integrated F origin of replication ( oriV ). This way Ts mutation in dnaA of chromosomal DNA is suppressed by F integration.

24. Drug resistant plasmids Most R plasmids have two contiguous segments of DNA: a) Resistance transfer factor(RTF) b) R determinant RTF carries genes regulating DNA replication & copy number, transfer genes & sometimes the genes for antibiotic resistance. In some strains & with some plasmids an RTF-R composite plasmid could not be stably maintained & are continually segregated. Plasmid amplification happens when both segments coexist together in large number of plasmids. This increases the size of plasmid & if any antibiotic is added to the medium of growth, this increase is observed prominently. Reversion to original size of plasmid occurs when antibiotic concentration is withdrawn. An interesting group of R plasmids are F-like R plasmids including R1, R6 & R100. Genetic analyses showed that F-like R plasmids consists of a large segments of F linked to a typical R determinant

25. Colicinogenic plasmid Plasmids which produce proteins called colicins that prevent growth of susceptible competing bacteria, are called Colicinogenic plasmids. Colicins interact with sensitive bacteria & inhibit one or more essential processes such as DNA replication,transcription , translation, or energy metabolism. Colicin production is detected by an assay similar to that for detecting phage where colicin producing cell if placed on a lawn of sensitive cells inhibit growth of nearby bacteria producing a clear area in the turbid layer known as a lacuna . Colicins are of two types: true colicins & defective phage particles (gene products from remnants of ancient defective prophages ) that resemble phage tail structure. Some colicins are inactive against a cell that contains a related col plasmid. This phenomenon is called immunity. Immunity is conferred by a small protein that binds larger colicin protein & also kills Col - cells. ColE1 is a colicinogenic plasmid that depends on compatible F plasmid for its conjugative mobilization.

26. Linear plasmids It is still a common belief that plasmids are circular. However, linear plasmids have been reported to exist more than a decade ago. Two types of linear plasmids are known. One type contains covalently closed ends and are commonly found in Borrelia , the causative agent of lyme disease. The other type is characterized by the covalent attachment of proteins at the 5' ends and exists in a number of bacterial genera including Streptomyces , Rhodococcus , Mycobacterium and Planobispora . Such plasmids like linear chromosomes face a primer problem whereby the extreme 5’end of lagging strand cannot be replicated by DNA polymerase as no primers exists upstream. This is not a problem of circular plasmids as upstream primer DNA exists. Such problems are thought to be solved in Borrelia linear plasmids by having telomeric sequences on their ends. Also CC ends have same inverted sequences, which allow the plasmids to be replicated till the end without losing DNA from the ends.

27. Uses of plasmids Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Disease Models - Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models.

28. Gene therapy- plasmid vectors are used for the insertion of therapeutic genes at pre- selected chromosomal target sites within the human genome. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. eg: insulin & antibiotics.