Plasmids in Bacteria: Properties, Types and Replication

In this article we will discuss about:- 1. General Properties of Plasmids 2. Types of Plasmids 3. Replication 4. Incompatibility 5. Library.

General Properties of Plasmids:

Plasmids are defined as extra-chromosomal genetic elements, occurring chiefly in bacteria and rarely in eukaryotic organisms. In bacteria, plasmids are circular double-stranded DNA molecules which contain genes controlling a wide variety of functions. In yeast (Saccharomyces cerevisiae) an RNA plasmid has been found.

Plasmids are self-replicating elements, yet they are largely dependent on the host cell for their reproduction, because they use the host cell replication machinery. The first plasmid to be discovered was the sex-factor or F plasmid (F stands for fertility) of E. coli K12. This plasmid confers the ability to an E. coli cell (F⁺) to conjugate with another lacking this plasmid (P cell). The F-plasmid can exist in two alternative states, viz. it can either remain free in the cell or it can be integrated into the E. coli chromosome. Plasmids with such property are known as episomes.

Generally, the bacterial plasmids are 1 to 5% of the chromosomal DNA in size. Plasmids vary widely in size. The smaller plasmids have molecular weights ranging between 4 to 5 x 10⁶ Daltons, while the larger ones have molecular weights of 25 to 95 x 10⁶ Daltons.

Plasmids not only vary in size, but also in copy number which denotes the number of copies of a specific plasmid in a cell. Copy number is discontinuously variable i.e. some plasmids generally the smaller ones — have a high copy number, while the larger plasmids have characteristically a low copy number. Those having a high copy number are known as relaxed plasmids and those having c low copy number are called stringent plasmids.

Whatever may be the copy number, plasmids are generally distributed equally in the daughter cell during cell division. Rarely, a plasmid-free cell may arise spontaneously at a frequency of about 1 in 10⁴-cells. Plasmid-free cells may also be produced artificially by the use of mutagens.

The process is commonly called curing of plasmids. Usually, the low-copy number large plasmids have one or two copies per cell and are easier to be cured. The smaller plasmids, in contrast, may have 10 to 100 copies per cell.

So far as the biological functions of plasmids are concerned, they are not indispensable constituents of the bacteria. This is proved by the fact that the bacteria cured of plasmids can grow normally without any difficulty. However, the genes carried on the plasmid DNA confer special properties to the host bacteria and such properties may become advantageous under special environmental conditions.

For example, bacteria carrying the R-plasmids (resistance plasmids) can survive when the environment contains inhibitory concentrations of one or more antibiotics. Obviously, the R-plasmid-less bacteria are destroyed under such conditions.

Another example is provided by the plasmids of some species of Pseudomonas which carry genes for production of enzymes catalyzing degradation of complex hydrocarbons. Bacteria carrying such plasmids are capable of using such unusual substrates for growth and, obviously, enjoy special advantage over others lacking them.

The F-plasmid gives the power to carry out a type of sexual reproduction to bacteria making it possible to exchange genetic materials leading to genetic recombination. Again, some bacteria, like E. coli, Pseudomonas, Lactobacillus etc. produce special type of proteins, called bacteriocins which are coded by plasmid genes. These proteins are able to kill other closely related bacteria and, thereby, they can eliminate competition for food and space.

Thus it is seen that even though plasmids are not absolutely essential for the life of bacteria under normal conditions of growth, their presence may become valuable and advantageous for the host under special conditions, or may even prove critical for survival as in case of the R-plasmids. The R-plasmids with the help of the resistance genes produce proteins which can inactivate or destroy specific antibiotics.

Besides the advantageous properties attributable to the plasmids, these extra-chromosomal genetic elements have played an important role in the development of recombinant DNA technology. In this technology, the plasmids are used as vectors for transferring a gene of interest from one organism to another organism. Such transfer of a gene is possible, not only from one bacterium to another, but also from an eukaryotic organism to a bacterium, or vice versa.

A segment of DNA containing the specific gene is isolated from a suitable donor and inserted by recombinant DNA technology into a plasmid. The recombinant plasmid is next introduced into a suitable host cell where the gene is expressed producing the gene product.

In this way, several human genes producing therapeutically important proteins have been introduced into bacteria. Also, some bacterial genes have been transferred to eukaryotic hosts, like plants, and some viral genes have been transferred to yeasts. In most of such gene transfers, plasmids play a key role as vectors or carriers.

Types of Plasmids:

(i) F-Plasmid:

The F-plasmid, also known as the fertility factor or sex-factor, determines the sex of E. coli bacteria. The cells containing this plasmid are designated as F⁺ and those without it as F^–. F⁺ bacteria are considered as male, because they can act as donor of not only the plasmid, but also chromosomal genes to the F^– cells which act as recipient and are, therefore, considered as female.

The process of transfer takes place by conjugation of the F⁺ cell with the F^– cell. The F-plasmid is a conjugative plasmid.

we know that a characteristic feature of the F-plasmid is that it can either remain as an independent entity replicating separately along with the chromosomal DNA, or it can be inserted into the chromosome as its integral part. When an F-plasmid is integrated into the E. coli chromosome, the bacterial cell changes from P to an Hfr-strain (high frequency of recombination).

There are many sites on the E. coli chromosome where the F-plasmid can be integrated. Depending on the site, each integration gives rise to a different Hfr-strain. In F⁺ x F^– conjugation, the plasmid alone is transmitted, but in Hfr x F^– conjugation, chromosomal genes are transmitted and rarely also the F-plasmid.

The F-plasmid is a large self-transmissible plasmid having a double-stranded circular DNA molecule. Its molecular weight is 63 x 10⁶ Daltons and it contains about genes controlling the transfer of the plasmid from the donor to the recipient. A mutation in any of the essential genes results in the loss of transmissibility of the plasmid.

Just as an F-plasmid can be integrated into the chromosome of the host cell, so it can though on rare occasions, be separated or excised from the E. coli chromosome in the free state to form the circular plasmid. It has been observed that the excision process is sometimes imperfect in the sense that some parts of the E. coli chromosome adjoining the linearly inserted F-plasmid are included in the excised F-DNA and at the same time, parts of the plasmid DNA are retained in the E. coli chromosome.

The F-plasmids containing parts of the chromosomal DNA are designated as F’-plasmids. When an F-plasmid loses some of its essential genes during the excision process, the plasmid is rendered incapable of independent existence and is, ultimately, eliminated during cell division.

When an F-plasmid is transmitted by conjugation to an F-recipient, it can transfer the chromosomal genes carried by it. Thereby, the recipient becomes diploid in respect of these transferred genes (because it now contains one copy of its own and another copy of the same gene transmitted by the F -plasmid). Thus, exchange of chromosomal genes may occur through F’-plasmids. This has been described as sex-duction.

(ii) R-Plasmids:

R-plasmids conferring resistance to various drugs individually or multiple resistance to several antibacterial agents were first discovered in Japan in the 1950s in the gastroenteritis-causing Shigella dysenteriae. Since then these plasmids have been found in E. coli and other enteric bacteria. Such plasmids have proved a great threat to the medical science.

The large R-plasmids having molecular weights ranging between 30 x 10⁶ Daltons are self- transmissible by conjugation with other bacteria. They are, therefore, conjugative plasmids, like the F-plasmid.

Smaller R-plasmids having molecular weights of about 5 to 6 x 10⁶ Daltons are non- transmissible. Most of the self-transmissible large plasmids like R100 of Shigella conferring multiple drug resistance are co-integrates of two DNA segments joined to each other by covalent linkage to form a single double-stranded circular molecule.

One DNA segment is called the resistance transfer factor (RTF), while the other segment contains the drug-resistance genes. The RTF is mainly involved in the transfer function of the R-plasmid and contains a number of genes (the transfer genes) and some others controlling replication of the plasmid in the host cell.

The resistance genes located in the other segment elaborate enzymes for destruction of the antibacterial drugs, like penicillins, streptomycin, chloramphenicol, tetracyclines, kanamycin, sulfonamides etc. (Fig. 9.89).

In some drug-resistant bacteria, such as Salmonella typhimurium strain 29, the resistance genes are located not in the same plasmid, but in separate plasmids of different size. This is sometimes known as plasmid aggregation.

The transposable elements that complex transposons may carry genes for drug resistance. Such elements can be integrated into plasmids giving rise to a drug-resistance plasmid. Thus, R plasmids may be made up of a collection of transposons, each of which may carry one or more genes for antibiotic resistance. For example, Tn 5 carrying a gene for kanamycin resistance may be inserted into the plasmid R100 of Shigella making the plasmid able to resist the antibiotic.

Besides drug resistance, plasmids may also make bacterial hosts resistant to the toxic effects of heavy metals. Plasmid-coded resistance to nickel, cobalt, mercury, arsenic and cadmium has been reported in different species belonging to the genera Pseudomonas, Escherichia, Salmonella and Staphylococcus.

(iii) Col-Plasmids:

The Col-plasmids are present in different strains of E. coli and they contain genes controlling synthesis of a class of proteins called colicines. Colicines are able to inhibit the growth of related bacteria which lack a Col-plasmid (Cor).

Several different types of Col-plasmids have been discovered, each of which produces colicines having a different mode of inhibition of susceptible bacteria. For example, Col B induces a damage of the cytoplasmic membrane of the target bacteria and Col E2 and Col E3 cause degradation of nucleic acids.

Like R-plasmids, Col-plasmids may be self-transmissible or non-self transmissible. Large Col plasmids, like Col I and Col V-K94 having molecular weights of 60 x 10⁶ Daltons or above are self- transmissible. They have a small copy number, usually 1 to 3 copies per cell. Small Col-plasmids, like Col-El, have molecular weight weighs of about 4 to 5 x 10⁶ Daltons.

They have a high copy number, usually 10 to 30 copies per cell. They are self-non-transmissible, but may be mobilized with the help of F-plasmid. This means that when an F⁺-cell contains also a Col El plasmid and conjugates with an F- cell, the Col El plasmid can be transferred to the recipient through the mating bridge constructed by the F-plasmid. Obviously, an F-ColEl⁺ cell is unable to mobilize the Col-plasmid to another cell, because it is unable to build a mating bridge.

In contrast, the large Col-plasmids are self-transmissible, because they have the genes for building the conjugation apparatus themselves and do not depend on the F-plasmid for transfer to other cells. Like F and large R-plasmids, the large Col-plasmids are also conjugative plasmids.

Colicins belong to a general class of proteins, called bacteriocins. Many bacteria have been found to elaborate bacteriocins which are able to kill other related or even unrelated bacteria. Such proteins are coded by genes present in bacteriocinogenic plasmids.

Bacteriocins produced by different bacteria are sometimes given different names, like pyocine produced by Pseudomonas aeruginosa, megasine elaborated by Bacillus megaterium, nisin by lactobacilli, etc. In general, bacteriocins exert their antibacterial action by binding to the cell wall of the target cells and by inhibiting one of the vital metabolic processes, like replication of nucleic acids, transcription, protein synthesis or energy metabolism.

Bacteriocins produced by enteric bacteria help to maintain a healthy ecological balance in the human colon. Other bacteriocins produced by bacteria under natural environmental conditions probably function by eliminating competitors. Nisin produced by lactic acid bacteria has been commercially used for preservation of food and dairy products.

(iv) Degradative Plasmids:

Degradation or dissimilation of organic compounds in course of mineralization is often controlled by plasmid-borne genes in many microorganisms. Such plasmids with genes coding for enzymes that catabolize complex organic molecules are known as degradative or dissimilation plasmids. For example, in species of Pseudomonas, both chromosomal and plasmid genes produce enzymes for break­down of complex compounds.

Some of the plasmid genes code for enzymes which degrade such unusual compounds like camphor, toluene, naphthalene, salicylate and complex hydrocarbons of crude petroleum. With the help of these enzymes, the bacteria can utilize these compounds as source of carbon and energy.

As a result, bacteria possessing such degradative plasmids stand a much better chance of survival under conditions where only such unusual compounds are available. Normal bacteria without such plasmid-coded enzymes would perish under similar conditions.

The capability of organisms carrying degradative plasmids to metabolize unusual diverse complex compounds suggests the possibility of employing them as means of bioremediation of the polluted environment. The development of genetic engineering techniques has encouraged scientists to develop genetically improved strains of bacteria containing plasmids capable of degradation of an array of complex compounds, such as those occurring in crude petroleum.

A synthetic strain of Pseudomonas has been developed by Anandamohan Chakraborty of the university of Illinois, USA offering prospects of practical use in removing oil-spills in the oceans, caused by leakage of crude petroleum from tankers. Oil-spills prove a great danger to marine life, both plants and animals.

(v) Ti-Plasmid of Agrobacterium:

Ti-plasmid is a tumour-inducing large extra-chromosomal double stranded circular DNA which is present in Agrobacterium tumefaciens, a plant-pathogenic bacterium causing the crown-gall disease in many dicotyledonous species. Crown-gall is a tumour produced at the collar region of plants by agrobacteria which possess the Ti-plasmid. Bacteria lacking the plasmid are non-virulent.

Ti-plasmid is about 200 kilo base-pair long circular DNA. Only a small part of this large molecule, a 30 kilo base-pair long fragment is responsible for tumour formation. This fragment is called the T-DNA (T stands for transformation). When Agrobacterium infects a susceptible host plant, the Ti-plasmid is released in the host cell and a copy of the T-DNA is integrated into the genome of the host plant.

The integrated T-DNA then stimulates cellular atrophy producing eventually a tumour, called a crown gall. The T-DNA insertion in plant host genome is the first instance of an inter-kingdom genetic exchange by natural means.

A notable feature of T-DNA is that once it is incorporated into the host genome, the presence of the pathogenic organism is no longer necessary for induction of tumour. Thus, a close parallelism with cancer induction in animal cell is observed. The T-DNA segment of the Ti-plasmid contains genes controlling synthesis of phytohormones, like indole acetic acid and cytokinins, as well as several other compounds, called opines. Opines, such as octopine and nopaline are used as growth substrates by agrobacteria.

The rest of the Ti-plasmid contains several genes controlling virulence (vir genes). These genes control T-DNA transfer to the host. Other genes of the plasmid control functions relating to bacterial conjugation, DNA replication and catabolism of the opines synthesised by gene products of the T-DNA segment.

The T-DNA acts as a mobile unit like a transposon, but it does not have a gene, like transposase to mediate its own mobilization . Its mobilization is effected by genes located in the Ti-plasmid, but outside T-DNA. The 30 kilo base long T-DNA is flanked on either side by 25 base pair imperfect direct repeats forming T-DNA borders.

The vir genes of Ti-plasmid are involved in the generation of a transferable copy of T-DNA and its transfer to plant cell through the cell membrane and the nuclear membrane, as well as through the bacterial and plant cell walls. T-DNA is transferred as a single-stranded copy.

The copy is separated from T-DNA segment, capped at the 5′-end by a protein coded by a vir gene (vir D2) and covered by a large number of protein molecules coded by another vir gene (vir E2). This T-complex is transported to the plant cell through a membrane pore produced by another vir gene. The T-complex (ss-DNA + proteins) is about 3.6 μm long and less than 2 nm thick.

A gross structure of the Ti-plasmid and generation of the T-complex have been diagrammatically represented in Fig. 9.90:

The ability of Agrobacterium tumefaciens to transfer its Ti-plasmid to many dicotyledonous plants (but not monocotyledonous ones) opened up the possibility of introducing foreign genes into the hosts using the Ti-plasmid as a vehicle (vector).

This has been practically employed to insert a gene of interest into the T-DNA segment by recombinant DNA technology. The tumour-inducing genes and other unnecessary genes of T-DNA are removed and replaced by the gene chosen for insertion. Several foreign genes have been introduced into a variety of hosts to produce transgenic plants.

Among the notable achievements are productions of transgenic plants resistant to the herbicide glyophosate and to feeding insects. Glyophosate resistance gene was isolated from Salmonella and the insect-resistance gene from Bacillus thuringiensis which synthesise an insecticidal protein. Another interesting achievement though not of practical significance was production of bioluminescent tomato plants by introducing the gene controlling bioluminescence in fire-fly.

(vi) Eukaryotic Plasmids:

Plasmids occur rarely in eukaryotic cells. Some plasmids have been found in yeast (Saccharomyces cerevisiae) and in several plants. The only RNA plasmid discovered till now has been found in yeast. It is a double-stranded RNA having a molecular weight of 15 x 10⁶ Daltons. It contains 10 genes including one coding for a bacteriocin-like protein. The protein can kill other yeast cells lacking the plasmid. This yeast plasmid has been designated as killer particle.

Yeast also contains small DNA plasmids with high copy number. They are located in the nucleus and like the chromosomal DNA are associated with basic proteins — histones. Some yeast DNA plasmids have been genetically engineered in such a way that they are capable of multiplication in both E. coli and yeast.

One such engineered yeast plasmid is Yep which can function as a shuttle vector. This plasmid has been used in transfer of useful genes from other organisms into yeast cells via E. coli for production of valuable therapeutically important proteins. A successful application of the Yep plasmid is the transfer of the gene coding the coat glycoprotein of hepatitis B virus to yeast.

The transgenic yeast can express the gene successfully with production of the viral glycoprotein. The glycoprotein has been used for preparation of hepatitis B vaccine for human application. Shuttle vectors are specially useful in transferring eukaryotic genes, because such genes are often not successfully expressed in bacterial hosts.

Besides yeast, DNA plasmids have been discovered in several plants, like maize and sorghum, as also in several fungi. These plasmids are made of usually linear double stranded DNA molecules where as all bacterial plasmids are circular.

Replication of Plasmids:

In the non-dividing plasmids, the double-stranded DNA exists as a right-handed super-helical coil having 400-600 base pairs per turn of the coil. During replication, the plasmids can multiply autonomously, although replication requires the host cell enzymes. That is why plasmids can multiply only within host cells.

Each plasmid has its own origin of replication. Some plasmids also have genes which code for proteins necessary for their own multiplication. This is proved by the fact that a temperature-sensitive mutant of F-plasmid (F⁺ts) is unable to replicate at 42°C, although it can function normally at 37°C.

Different aspects of plasmid replication are briefly discussed below:

(i) Non-Transmissible Plasmids:

Replication of plasmid DNA starts at the site of origin and may proceed either bi-directionally as in case of bacterial chromosome, or may proceed unidirectionally depending on the nature of plasmid. In bidirectional replication, replication terminates when the two replication forks meet each other. In unidirectional replication, termination occurs when the replication fork reaches the site of origin. In both cases, the circularity of the plasmid DNA is maintained throughout the process.

(ii) Self-Transmissible Plasmids:

In case of conjugative plasmids, like F-plasmid or R-plasmid, replication occurs by the rolling- circle model. The supercoiled DNA undergoes a nick in one of the strand resulting in relaxation of the super coiled state to form an open circle. The enzyme catalyzing the nick remains attached to the 5′-P end of the relaxed molecules. Such a single-stranded nick becomes necessary for transfer of a copy of the plasmid during conjugation to the mating partner.

By rolling circle replication, the donor cell retains its double-stranded plasmid, while a single-stranded copy is transferred through the mating bridge to the recipient cell, where a complimentary strand is synthesized and ligated to form a double-stranded copy of the plasmid. Replication of an F-plasmid is diagrammatically shown in Fig. 9.91.

(iii) Control of Copy Number:

The large plasmids are characterized by low copy number (one to few) and small plasmids by high copy number (10 to 100). The copy number is controlled by an inhibitor coded by the plasmid DNA itself. The inhibitor concentration in the bacterial cell determines the rate of initiation of plasmid replication.

When a cell containing two large plasmids divides to produce two daughter cells, each having one plasmid, the inhibitor concentration in these cells is the same as that of the mother cell. Now, the daughter cells grow in size to attain maturity resulting in lowering of the inhibitor concentration in the cytoplasm.

As a consequence, DNA synthesis is initiated in the plasmid leading to its replication producing two copies. As each plasmid copy possesses an inhibitor gene, the production of inhibitor doubles and the inhibitor concentration becomes high enough to stop plasmid DNA synthesis and further replication. Thus, the copy number is restricted to two per cell.

A similar mechanism of control of copy member is believed to operate in case of high copy number plasmids also. However, in this case, the inhibitor concentration must reach a higher threshold level to stop initiation of plasmid DNA synthesis in comparison to that of low copy number plasmids.

(iv) 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 amplication becomes a useful tool, because of high plasmid DNA concentration in the total cellular DNA.

(v) Transfer of Non-Self Transmissible Plasmids:

There are some plasmids which do not possess genes for self-transmission, but can be transferred to other cells with the help of a self-transmissible plasmid when both plasmids occur in the same cell. They are known as mobilizable plasmids.

Such plasmids possess genes for proteins needed for nicking its own DNA at the site of origin of replication, but lack in genes needed for building the conjugation tube. When they coexist with a self-transmissible plasmid, like F or R, the latter can build the mating bridge through which a copy of the mobilizable plasmid produced by rolling-circle replication is transferred to a recipient cell (Fig. 9.92).

A different type of mobilization occurs when a donor cell having a self-transmissible plasmid conjugates with a recipient having a mobilizable plasmid. In this type of conjugation, both the donor and the recipient acquire a copy of both types of plasmids by a process of retro transfer. First, the self- transmissible plasmid replicates by rolling-circle model and a single-stranded copy is transferred through the mating bridge to the recipient, where it forms a complimentary strand leading to the formation of a copy of the self-transmissible plasmid in the usual way.

The mobilizable plasmid in the recipient cell then replicates and a single-stranded copy is transferred to the other cell which now acts as the recipient of the mobilizable plasmid. Finally, the two cells separate and each has a copy of the self- transmissible plasmid and a copy of the mobilizable plasmid (Fig. 9.93).

Incompatibility of Plasmids:

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.

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.

One mechanism by which a plasmid already resident in a cell prevents the entry of a second similar plasmid into the same cell is by surface exclusion. For example, an F-plasmid of E. coli does not allow entry of another F-plasmid by inhibiting it from leaving the cell where it is already located. The effect is mediated at the surface of the cell whereby the F-DNA cannot come out of the cell.

A different mechanism operates when a cell already has two closely related plasmids, say X and Y. We know that the copy number of plasmids is controlled by specific inhibitors coded by the plasmid itself.

As X and Y are two closely related plasmids, it would be expected that their inhibitors would also be closely similar and that replication of both the plasmids would be regulated by the inhibitor produced either by X or Y.

During replication, X and Y may be selected at random, so that, during first replication of the plasmid, a cell initially containing one copy of each plasmid may produce two copies of either X or Y, so that the cell has now two copies of either X or Y and one copy of the un-replicated plasmid i.e. 2X + Y or X + 2Y. In the second round of plasmid replication, each cell will contain 4 plasmids, but depending on which plasmid is replicated, the combination may be X + 3Y, 2x + 2Y or 3X + Y.

Now the cell divides to produce two daughter cells, each with 2 plasmids and the plasmid combinations of the daughter cells may be X + X, X + Y or Y + Y. Thus the probability of progeny cells having either two X plasmids or two Y plasmids is equal to those having two different plasmids i.e. X + Y. In other words, the probability of elimination of one plasmid is 50%. Such probability increases with more cell generations.

The events leading to plasmid elimination are shown in Fig. 9.94:

Plasmid Library:

A plasmid library is a gene library which contains a collection of bacterial cultures, each of which contains a plasmid, but plasmid of one culture differs from that of another in having a separate DNA fragment of a genome of an organism. The total genome isolated from an organism is fragmented and the fragments are inserted (cloned) separately into individual plasmids.

These recombinant plasmids are then introduced into suitable host bacteria. Thus each bacterial culture contains a plasmid with a fragment. The total collection of cultures would be expected to contain the entire genome of an organism and would constitute a gene library of the particular organism. This is schematically represented in Fig. 9.95.