BSc 2nd Year Microbiology Microbial Genetics Notes Study Material

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BSc Microbiology Microbial Genetics Notes Study Material

During last twenty-five years or so, there has been a dramatic increase in our knowledge of the mechanisms that determine the characteristics of the microorganisms. What we know indeed of the structure and organisation of DNA, has been possible only through the work on microbial genetics, particularly of bacteria and fungi. Microbes have proved ideal organisms of study in molecular biology. We now understand the functioning of genome at molecular level and making efforts to manipulate cells for improving their specific desired properties. This has resulted into a new phase of exploiting bacteria and fungi in industry i.e. in biotechnology.(BSc Microbiology Microbial Genetics Notes Study Material)

The hereditary characteristics are coded on DNA, in discrete units – genes. During cell division, DNA replication occurs, for which decoding of the genes is carried out in two steps called transcription and translation giving rise to the polymers of RNA and protein respectively. Thus information transfer in cells almost always occurs in the way shown in Figure. The processes of transcription and translation are diagrammatically shown in Figures respectively.

Gene

The important relationship between DNA and the characteristics of an organism was derived from studies of both fungi (Neurospora crassa) and bacteria (Pneumococcus, E. coli). The main finding that genes act by controlling the specificity of protein synthesis was obtained from work with mutants of the fungus Neurospora crassa, that led to propose that

1 gene = 1 enzyme

Bacteria, particularly E.coli have been used to define this relationship more precisely. Thus:

1 gene (or cistron) = 1 polypeptide

This definition reflects the the discovery that not all polypeptides are enzymes. Some play a structural role (as flagellin), and others are regulatory (as the catabolite repressor protein, CRP).

Transcription is the copying of DNA to give intermediary molecule, the mRNA, carried out by RNA polymerase. In translation the RNA is translated into a sequence of amino acids that make the polypeptide. This requires rRNA and TRNA. Translation takes place in ribosome.

Control of Transcription

There are two types of genes: structural and regulatory.

1. Structural genes. These are majority of genes, which produce specific polypeptide which may act directly or in combination with other peptides to form enzymes or structural proteins. Such genes are called structural genes. In bacteria these genes usually occur as a cluster on the chromosome, the region being called an operon. Such multigenic operons produce single molecule of mRNA initiated by the binding of RNA polymerase to a promoter sequence at one end of the operon.
2. Regulatory genes. These are situated immediately adjacent to the promoter sequence, sometimes even overlapping with it and are the regulatory sequences which control the initiation of transcription. These sequences are the site of action of polypeptides produced by regulatory genes.

There are basically two types of control of transcription, negative and positive.

(a) Negative control. The basic action of these regulatory polypeptides is to combine within or close to the promoter at a region known as the operator. Binding of the regulatory protein to the operator prevents the binding of RNA polymerase to the promoter. Synthesis of RNA is thus inhibited and no synthesis of protein coded for by the operon occurs. In other words, control is essentially by competition between the RNA polymerase and the regulatory protein for the promoter.(BSc Microbiology Microbial Genetics Notes Study Material)

(b) Positive control. This type of control has been recognised only during last ten years. Here the initiation of transcription is dependent upon the binding of the regulator to the promoter. Examples are many, as (i) the catabolite repressor protein (CRP) where CRP forms an active complex with cyclic AMP (CAMP), which acts on many different operons to increase the rate of their transcription (over 200 genes are regulated by CRP-CAMP complex in E. coli). This is called global regulatory system as it affects genes of many different metabolic systems. (ii) specific regulator, the one which is specific to only one operon or group of operons. When a number of operons are under the control of a single specific regulator it is termed a regulon. An example of this type is the arabinose regulation of E. coli.(BSc Microbiology Microbial Genetics Notes Study Material)

Regulation of gene expression usually requires the presence of another molecule other than the protein produced by the regulator gene. This is usually either the inducer or the repressor depending on its mode of action and the processes of regulation are known as induction or repression respectively. Both can be found in either negative or positive regulatory systems.

1. Induction. The inducer specifically combines with the regulatory protein rendering it either effective at binding to the regulatory sequence and thus stimulating transcription (positive regulation) or incapable of acting on the operator sequence and thereby switching on induced protein synthesis (negative regulation).
2. Repression. Binding of the repressor to the regulatory molecule either prevents interaction of the positive regulator with the regulatory sequence or increases the affinity of the negative regulator for the operator sequence (negative regulation) and thus stops transcription.

Mutations in Microorganisms

Mutation is a natural phenomenon resulting into variations within any population of cells. This is a change in the DNA sequence of a gene and is said to lead to a change in the genotype of the organism. Various types of mutants are found in microorganisms. These are as follows:

1. Auxotrophic mutants. Many microbes such as E. coli can grow on a medium containing a single carbon and energy source. They are called prototrophs. If a mutation occurs which results in the loss of the ability to synthesise an essential metabolite such as an amino acid or a growth factor, it will express itself in a nutritional requirement for that substance. Such mutants are called auxotrophs. These proved much useful in establishment of the relationship between genes and enzymes, as markers for genetic experiments in construction of genetic maps etc.
2. Resistant mutants. Bacteria may develop resistance to antibiotics and phages spontaneously through a range of mechanisms, e.g. loss of cell surface components which act as phage receptors, and acquisition of enzymes able to metabolise the antibiotic.
3. Metabolic mutants. These mutants have lost the ability to use a particular carbon source and are usually affected either in transport or metabolism. The mutants which have lost the capacity to make specific cell components as the capsular polysaccharides and some those which exhibit altered colonial shape (arising from mutations affecting cell wall synthesis) also belong to this category.
4. Regulatory mutants. In these mutants, mutation affects either the regulatory region of the promoter of the gene or the activity of a regulatory protein.

The above mentioned types of mutation are common in nature and the laboratory. This classification of mutations is based on their effects on the genotype of the cell. There are also other different kind of mutations, which are shown in Table.

Spontaneous mutations. They take place in nature without human intervention or identifiable cause. On an average in a colony of one billion (10%) bacteria, at least one mutant may be present. Since 1976, a penicillin – resistant strain of Neisseria gonorrhoeae (gonorrhea – causing bacterium) has been emerging in human populations. Antibiotic – resistant serotypes of Salmonella are also developing in nature.(BSc Microbiology Microbial Genetics Notes Study Material)

Induced mutations. These are the mutations in which cause can be identified. More often they result from planned experiments, in which microbes are subjected to physical or chemical agents. The most common agents used to inducing mutations (mutagens) are, ultraviolet light, nitrous acid and several base analogs (the chemicals closely related to nitrogenous bases of DNA). One base analog is 5-bromouracial. Other mutagens include, benzopyrene (in industrial soot and smoke), aflatoxin (a fungal toxin found in animal products and foods) etc.(BSc Microbiology Microbial Genetics Notes Study Material)

Transposable Genetic Elements

Transposable genetic elements (TGE) are fragments of DNA. These may cause totally a different type of mutations in nature. Two types of TGE are known: insertion sequences and (ii) transposons.

Insertion sequences. They are small segments of DNA with about 1000 base pairs, found at one or more sites on the bacterial chromosome and appear to have no specific genetic information except their ability to insert onto a chromosome. Insertion sequences form copies of themselves, and the copies move from normal position into areas of gene activity. Here they interrupt the coding sequence.(BSc Microbiology Microbial Genetics Notes Study Material)

Transposons. They are the so-called jumping genes for which Barbara Clintock won the 1983 Nobel Prize in Physiology or Medicine. Earlier in 1951, her work on Indian corn plants revealed that genes may not be always fixed elements and found in the same position on the same chromosome. She could describe that genes apparently moved from one chromosome to another. She watched over the changes in colour in her plants and the pigment genes responsible for colour change appeared to be switched on or off in particular generations. More remarkable was that these switches occurred in later generations at different places along the same chromosome. In modern molecular genetics, these controlling elements are a two-element system: an activator gene and the other a dissociation gene. The activator gene can direct a dissociation gene to jump along the arm of the ninth chromosome in maize plants where colour is regulated.

Jumping gene is identical to the transposon found in bacteria. Transposons, first identified and named in 1974 by British Microbiologists, R.W. Hedges and A.E. Jacobs, are larger than insertion sequences and carry information for protein synthesis. They contain genes for antibiotic resistance. They may move from plasmid to plasmid, from plasmid to chromosome or from chromosome to plasmid. The movement of transposons is nonreciprocal i.e. an element moves away from its location and nothing takes its place. This contrasts with insertion sequences where copies move.

Overlapping genes

For years scientists assumed that a mutation gene affected the production of single protein. However, Frederick Sanger’s (1977) work indicated otherwise. He determined the entire nucleotide sequence of a viral DNA molecule and mapped its, 5374 bases. Analysis of the DNA by Sanger’s coworkers showed that at least four of the genes were overlapping. This showed that an individual triplet code for two different amino acids. In 1958 Sanger won the Nobel Prize in chemistry for his sequencing of the amino acids in insulin. In 1980 he shared a second Nobel Prize in chemistry for mapping the bases of the viral DNA.

Ames test

It was seen that about 90% of the agents that cause cancer in humans also cause mutations in bacteria. Working on these lines, Bruce Ames at University of California, USA developed a procedure to determine whether a chemical can induce a bacterial mutation and thus be a potential agent of cancer. The procedure, Ames test is relatively inexpensive, accurate and rapid. A histidine – requiring strain of Salmonella typhimurium is inoculated into a plate containing the culture medium lacking histidine. Normally the Salmonella strain will not grow as genes for histidine-synthesis are lacking. Now the potential cancer agent is added to the medium and plate is incubated. If bacterial colony appears, one may conclude that the agent mutated the bacterial genes to code for enzymes needed for histidine synthesis. The agent is, therefore, a possible cause of cancer. If bacterial colonies fail to appear, no mutation takes place. However, the test works only within specified limits.

Enrichment and replica plating

Mutation rates can be increased by the use of mutagens like UV-light, ionizing radiations and chemicals like nitrous acid, nucleotide analogues etc. Methods have been developed for isolation of a particular desired type of mutant. There may be some difficulty in finding a suitable method to enrich specifically for the desired mutant and to recognise and isolate it when it may still be only a small proportion of the irradiated population. Direct selection can only be achieved when the mutation gives rise to resistance to a chemical or virus.

Enrichment step is very useful in increasing the percentage of cells carrying the mutated gene. One such method is penicillin (or more frequently ampicillin) enrichment. The cells are first grown in normal growth conditions (as those used for parent cells). The mutagenized cells are then transferred to an environment in which the desired mutant can not grow and after a short time (to allow the growth of mutant to cease) ampicillin is added to kill any cells which are still able to grow. The survivors of such a treatment are enriched for the desired mutant.

Nutritional and various other types of mutants are often detected by replica plating technique, developed by Joshua and Esther Lederberg in 1952 in order to provide direct evidence for the existence of preexisting mutations. Their actual experiment concerned with replicating master plates of sensitive cells to two or more plates containing either streptomycin or phage. When the replicas were grown, they were compared to the location of colonies on the master plate and any resistant colonies that appeared the same position on all the replica plates were marked. The area of master plate corresponding to the marked areas was cut out and bacteria on it resuspended in liquid medium.

This method has been applied in numerous experiments to identity occurrence of mutations. Many of the biochemical pathways in microbes were elucidated in this way by using nutritional mutants. Replica plating allows the observation of microbes under a series of growth conditions. In replica plating, a piece of sterile velvet is touched to the surface of an agar plate containing surface bacterial colonies. The fibres of velvet act as fine inoculating needles, picking up the bacterial cells from the surface of this master plate. The velvet with its attached microbes is then touched to the surface of a sterile agar plate, inoculating it.

In this manner, microbes can be repeatedly stamped onto media of differing composition. The distribution of microbial colonies should be exactly replicated on cach plate unless the colonies represent different genetic strains. Should a colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains.(BSc Microbiology Microbial Genetics Notes Study Material)

This technique was specifically developed to answer an important and controversial question-did mutations occur spontaneously, or directed by the selective agent? For example in case of penicillin resistance, does penicillin direct the mutation or does it simply select out a naturally occurring spontaneous mutant? It was shown by statistical analysis of mutants that the process is indeed a spontaneous one. This method also allows the isolation of penicillin-resistant mutants without ever exposing the cells directly to penicillin.

Mutations and adaptability

The selection of spontaneous mutants by the environment and the exchange plasmids (we shall refer to these DNA molecules later in this chapter) between organisms can occur more quickly in microorganisms. This may result into a rapid change in the genotype of the dominant organism in a population of cells. As compared with higher organisms, the mutation rates in microbes are higher, and spread newly acquired characteristics occurs at faster rate. This is due to the following:

(1) Most microbes are haploid, thus mutations cannot be masked by allelie gene.

(2) They are unicellular, so many mutated cells can give rise to a new evolutionary line.

(3) Microbes can occur in high densities in restricted environments, where a mutant may have a selective advantage.

(4) Microbes grow very rapidly.

(5) Plasmid transfer is common, thus new traits do not have to be acquired by all organisms by a long process of mutation and evolution.

Due to above said characteristics, microbes, particularly prokaryotes, are able to adapt themselves to a wide variety of extreme environments.

Recombination in Prokaryotes

We have seen that genetic changes due to mutations can result in the acquisition of new biological characteristics and thereby allow evolutionary change. However, evolution of the fittest organism in a particular environment can be enhanced if transfer of genes between organisms is made possible genetic recombination. As compared with eukaryotes, where sexual recombination is of ordered nature, in prokaryotes the process is less well developed. It does not involve a true fusion of male and female gametes to produce a diploid zygote Instead there is transfer of only some genes from the donor cell to produce a partial diploid. This is followed by recombination to restore the haploid state. There are three mechanisms by which these DNA fragments can pass from a donor to a recipient cell (i) transformation, (ii) transduction and (iii) conjugation.

[I] Transformation

Transformation was discovered by an English bacteriologist, Frederick Griffith in 1928, who made a series of experiments with laboratory mice and two types of pneumonia-causing bacterium, Diplococcus pneumoniae. This bacterium has two types of strains. One type has smooth (S), capsulated cells, whereas another type has rough (R) noncapsulated cells. The disease is caused by smooth-type of cells only i.e. smooth-type cells are pathogenic (virulent) whereas rough type cells harmless or nonpathogenic (avirulent). The experiments conducted by him are illustrated. As shown in the figure, when live, harmless (rough type) cells were injected in the body of mice, the animal remained healthy. The injection of dead, pathogenic (smooth-type) cells into the body of mice also did not cause any disease. In a classic experiment, Griffith mixed live, harmless (rough type) cells with the dead remains of pathogenic (smooth-type) cells, and injected the mixture into the laboratory mice. The live cocci taken in the mixture were uncapsulated and formed rough colonies (R) on agar. The dead cocci taken in the mixture originally had a capsule and were taken from smooth (S) colonies on agar.

To Griffith’s surprise, the mice developed pneumonia and died. On autopsy (examination of tissue of dead animal), he isolated live, capsulated cells that formed smooth colonies on agar. Apparently the live, harmless rough cocci had been transformed in the mice into live, pathogenic, smooth cocci.

A rough to smooth conversion (R → S) had been accomplished. Five years later, James L. Alloway of Rockefeller Institute confirmed Griffith’s work using fragments from the dead smooth-type cells to transform the rough-type cells. In 1944, Oswald T. Avery, Colin M. MacLeod, and Maclyn N. McCarty, also of Rockefeller Institute found that deoxyribose-nucleic acid (DNA) isolated from the fragments could induce the transformation. At that time, DNA was an obscure chemical with little significance. The work of Avery, MacLeod and McCarty helped bring it to the force. Their experiments were the first proof that in living organism genetic matter is DNA. The possible mechanism of transformation is shown in Figure. Though it takes place in less than 1% of a population, transformation is an important method of recombination in bacteria. A number of donor cells break apart and an explosive release and fragmentation of DNA follows. A segment of double-stranded DNA containing about 10-20 genes then passes through the cell wall and membrane of a recipient cell. Only a few competent recipient cells can take up the DNA. After entry into cell, an enzyme dissolves one strand of DNA leaving the second strand to be incorporated. This strand then displaces a segment from a strand of the recipient’s DNA. The displaced DNA is dissolved by another enzyme in the cell. The cell is now transformed. It will display its own traits as well as those coded by the new DNA.(BSc Microbiology Microbial Genetics Notes Study Material)

Transformation may also take place by the incorporation of plasmids to competent cells. In this case, no DNA is displaced. Rather, the plasmid adds genes to those already in the cell and multiplies along with the cell.

Since the 1940’s, transformation has also been demonstrated in species of Neisseria, Bacillus. Haemophilus, Azotobacter and Streptococcus. The process involves the transfer of DNA from the fragments of donor cells into the cytoplasm of a live recipient cell. Sections of single-stranded or double-stranded DNA may be taken up but only a single strand will align with the bacterial chromosome and becomes incorporated into it.

Transformations in bacteria have been observed in the ability to form a capsule, a drug resistance and pathogenicity, and in nutritional patterns. Transformations are not common, however, because the large fragments of DNA molecule can not pass through the recipient’s cell wall or membrance. In nature, transformation may increase the pathogenicity of an organism.

In cells of some genera of bacteria, DNA binds to the cell surface before uptake. However, this is restricted only to some genera. This problem has been overcome in both, pro- and eukaryotes by the removal of the cell wall to form protoplasts. Addition of a compound-fusinogen allows the protoplasts to fuse and mixing of DNA of two or more cells for recombination. This technique is known as protoplast fusion. When DNA is included in the incubation mixture transformation occurs simultaneously with fusion and thus small fragments of DNA e.g. plasmids, can be introduced into the cells. Protoplast fusion can also be managed between members of different species by mixing their protoplasts in the presence of a fusinogen. In this way genetic exchange can be achieved between organisms for which no other gene transfer system is known to exist.

[II] Conjugation

It involves a transfer of genetic material from one living cell to another during a period of contact. Whereas in both transformation and transduction any cell can act as a donor or a recipient, in conjugation some are donor of DNA while others are recipients. This process was first postulated by Joshua Lederberg and Edward Tatum in a series of experiments in 1946. Two strains of Escherichia coli were used. One was unable to synthesize an essential compound A whereas the owner could not synthesize an essential compound B. Neither strain was able to grow in a culture medium lacking both, compound A and compound B because each strain lacked an important enzyme system.

Lederberg and Tatum mixed the cells of two strains, and after a short incubation time, placed samples on the medium lacking both A and B. Surprisingly, bacterial growth appeared on this medium. When a few colonies appeared, it was first thought that transformation had occurred. However, it was shown experimentally that a physical cell contact was necessary for the recombination to take place. Apparently, the genes for synthesis of compounds A and B passed between the cells and yielded a recombined chromosome that could produce both the missing compounds. Their experiments are illustrated in Figure. Lederberg and Tatum shared the Nobel Prize of 1958 for their work in bacterial genetics.

Later experiments by Francois Jacob and Elie L. Wollman established that bacteria were of two mating types. Certain male types or F⁺ or donor cells, were those that donated some their DNA, whereas female type or F^– or recipient cells, were the recipient of the genes. It was noted that recipient cells would rapidly become donor cells when certain small amounts of DNA were passed to a recipient cell. This eventually led William Hayes to discover genetic factors, called fertility factors or F-factors, in the cytoplasm of the donor cell (male) apart from the chromosome. These are also called sex-factors or F-plasmids. In contemporary microbiology, the F-factors are known to be types of plasmids. They are double stranded loops of DNA, apart from the chromosome.

The factors contain about 20 genes, most of which are associated with conjugation. One function of these genes is to form a conjugation bridge or sex pilus, between donor and recipient cells. The F-factor then prepares for replication by the rolling circle mechanism. There is no complete process of replication but as in replication, the two strands begin to separate from each other and a single strand of the factor passes through the sex pilus to the recipient. When it arrives, enzymes synthesise a complementary strand and a double helix forms again. The double helix bends into a loop and the conversion from F to F⁺ cell completed. Meanwhile back in the donor cell, a new strand of DNA forms to complement the leftover strand of the factor. The word episome is commonly applied to plasmids that function in conjugation processes.

High-frequency of recombination. In the above mentioned process we have seen that the transfer of F-factors involved no activity of the bacterial chromosome. Therefore the recipient cell does not acquire new genes other than those on the F-factor. However, there exists in bacteria a type of conjugation that accounts for the passage of chromosomal material. Strains of bacteria that display this ability are called high frequency of recombination, or Hfr strains. Such strains were discovered in the 1950s by William Hayes in E. coli. Such strains developed when a male (F⁺) mutated to a super-male which showed a high frequency of recombination with a female (F^–), hence the mutant called Hfr.

In Hfr strains, the F-factor attaches to the chromosome. This integration is a rare event and requires that an insertion sequence be present on the chromosome to recognise the F-factor. At the point of attachment, the chromosome opens and a copy of one strand is made by the rolling circle mechanism. A portion of single-stranded DNA then passes via the sex pllus into the recipient cell. Here it joins the chromosome as in transformation. The first genes to enter the recipient are F-factor genes, but these are not the ones that control the donor state. Instead, the last segments to enter the recipient are the F-factor genes for the donor state. These rarely enter the recipient, however, because conjugation is often interrupted by such things as broken pili. Thus the F^– cell usually remains a recipient, but with some new genes acquired from the donor. In certain cases the F-factor is indeed transferred to the recipient. When this happens the factor usually detaches from the recipient’s chromosome and enzymes synthesise a strand of complementary DNA. The factor now forms a loop to assume an existence as a plasmid, and the recipient becomes a donor.

Sexduction. On occasion, the F-factor breaks free from the chromosome of an Hfr cell and resumes an independent status. The Hfr cell then reverts to an F⁺ cell. Sometimes when the F-factor leaves the chromosome, it takes along a segment of chromosomal DNA. The factor with its extra DNA is now called an F factor (pronounced F-prime). When the F factor is transferred during a subsequent the recipient acquires chromosomal genes from the donor.

This process is known as sexduction. It results in a recipient with its own genes for a particular process as well as additional genes from the donor for that same process. In the genetic sense, the recipient is a partially diploid organism since there are two genes for a given function. Conjugation has been demonstrated among various generia of bacteria in contrast to transformation which appears to occur only among cells of the same species. This has great significance in transfer of antibiotic resistance genes carried on plasmids. When the plasmids are F-factors, the transfer occurs readily. Moreover, when the genes are attached to transposons, the transposons may “jump” from ordinary plasmids to F-factors, after which transfer may occur, Conjugation may also occur in Gram-positive bacteria as Streptococcus mutans. Only plasmids especially those carrying genes for antibiotic resistance are involved in this species. Similar observations have been made in Bacteroides and Clostridium species. Till now mechanism of plasmid transfer in Gram-positive bacteria is poorly understood and chromosomal transfer is yet to be demonstrated.

Plasmids. Continuing research in bacterial conjugation revealed that several other genetic traits other than chromosome were present. The genes for the production of some toxins, for the production of pilus and for the antibiotic resistance were found on DNA fragments in the cytoplasm. The word plasmid was coined for these fragments independent of chromosome. Plasmids are small circular DNA molecules which replicate in cells independently of the chromosome. Such fragments have been termed by different authors, as sex factors, conjugons, extrachromosomal replicons and transfer factors. For instance, the genes responsible for transfer of resistance to some antibiotics are called resistance transfer factors. Those responsible for transfer of the property of producing colicins (antibiotics type substances lethal to closely related strain or species of the bacteria producing them) are called the colicin factors (Cf). The plasmids normally contain only about 2 per cent of the total genetic information, and multiply independently of the chromosome. They pass quickly from cell to cell as does the F-factor (fertility factor) and are found to be a critical factor in transferable drug resistance (TDR). Thus by genetic recombination, a pathogenic um may acquire the genes (for resistance to a particular drug) from a harmless (non-pathogenic) organism. For instance 1976 saw the emergence of penicillin-resistant gonorrhea organisms, and multiple resistance in pneumonia cocci were located in 1981. The plasmids are not essential to the cell growth and may be lost without any harm to the cell. Those plasmids that attach themselves to the chromosome are called episomes. Thus, the F-factor in Hfr cells is considered an episome. The study on plasmids was pioneered by the Stanford University microbiologist, Stanley Cohen who later utilised them in genetic engineering experiments. Plasmids in fact lie at the core of genetic engineering, which offers the potential to increase the range of antibiotics and to increase the availability of any compound in short supply e.g. insulin.

[III] Transduction

This was discovered in 1952 by Joshua Lederberg and Norton Zinder during their search for evidence of conjugation in Salmonella typhimurium. Recombination by this method requires a virus (bacteriophage) as an agent to carry genes from one bacterium to the next. Bacteriophage interacts with bacterial cells by a

complex process shown in Figure. The phage first attaches to a receptor site on the bacterial cell wall, after which nucleic acid passes into the cytoplasm of the host cell.(BSc Microbiology Microbial Genetics Notes Study Material)

At this point, either of two events may occur. In the first alternative, the nucleic acid codes for proteins that will form new virus particles identical to the original phage. Components are drawn from the host cell, and the metabolism of the latter is interrupted to such an extent that after some minutes the cell bursts apart to release new bacteriophages. This is the lytic cycle, from lyse means to break, and the phage that causes the lytic cycle is called a virulent phage.

In the second alternative, the phage does not cause the lysis of bacterium. Instead the phage nucleic acid codes for a substance called a repressor protein. This protein prevents the virus from directing the production of materials necessary for replication. Rather than forming new virus particles, the DNA may exist as a fragment of DNA in the host cytoplasm in a form called the prophage. It may exist as a fragment of DNA outside the chromosome, essentially as a plasmid, or it may attach itself to the chromosome as the F factor does in Hfr strains. This nonreplicating phage is called a temperate phage and the DNA fragment is known as prophage. The bacterium that carries the prophage is said to be lysogenic, and the phenomenon where the bacterium and phage DNA coexist is called lysogeny.

The bacterium may remain lysogenic for many generations during which time the viral DNA replicates together with the bacterium. However, at some point in the future, the phage stops coding the repressor protein, and the lytic cycle will begin.

The viral DNA that was attached to the chromosome will now break free and direct the synthesis of those proteins that will yield new viruses. In detaching, however, the viral DNA may carry with it a few bacterial genes from the chromosome. The genes are then replicated along with the viral DNA and they become part of the new phage particles. When the latter are released, copies of the genes are carried along. As the cycle repeats during the next infection, phage DNA enters the new bacterial cells and inserts onto a new chromosome. However, copies of the original bacterial genes are included, and the bacterium becomes transduced. The bacterial cell now contains its own genes plus several from the original cell. This type of transduction is called specialised transduction, because specific genes are removed from the bacterial chromosome, depending upon where the viral DNA was attached. This occurs in lambda phage. The removal of genes, however, is thought to be an extremely rare event. Another type of transduction, generalised transduction is a more common event.

It is mediated by the prophages that have remained in the cytoplasm as plasmids that are not attached to the chromosome. This occurs in Pl phage and many others. The viral DNA lies in the cytoplasm and produces copies of itself for new phage particles. In doing so it may accidentally incorporate small chromosomal segments of bacterial DNA and incorporates these to its own DNA. Some phages may accidentally package only bacterial DNA. In most cases, normal viruses will be liberated from the cell. Occassionally, a virus contains several bacterial genes acquired in the chromosomal segments. If such a virus infects a new cell, whereupon they will attach to the chromosome and transduce the cell as lysogeny is established. In generalised transduction, the viral DNA enters the lyric cycle and forms new virus particles. However, tiny fragments of bacterial chromosome are sometimes incorporated into the DNA of the new viruses or may occasionally replace the viral DNA. This is a random occurrence that may involve any of the bacterial genes, hence the name generalised transduction. Perhaps one phage in a thousand contains bacterial DNA. All bacterial genes are equally available to be picked up by the phage DNA. When the viral particles are release during lysis, the genes are carried along, and on subsequent infection, the genes enter the cytoplasm of the new host cell where they will now function.

The phenomenon of lysogeny is well established in modern microbiology. Diphtheria organisms are known to contain bacteriophages that code for the toxin produced during disease. Herpes simplex viruses remain for many years as prophages in the cytoplasm of the body cells, expressing themselves at long intervals. Certain viruses are known to attach to human chromosomes, transforming the cells to tumour cells.

Recombination in Eukaryotes

Unlike prokaryotes, in eukaryotes the genes are organised onto several chromosomes which are present in the nucleus. The number of chromosomes is species-specific and is a stable character of that species. Genetic recombination here is essentially a sexual process involving recombination during meiosis leading to the formation of male and female haploid gametes. Fusion of gametes results in the new types of diploids. However, in the fungi, there is some complexity. Unlike most eukaryotes, some only exist in the haploid state. Consider the life cycle of two fungi, Aspergillus nidulans and Saccharomyces cerevisiae. In A. nidulans, haploid mycelia of different strains fuse and form fruit body. In this fruit body there is karyogamy and meiosis. During meiosis the chromosomes from the two nuclei pair up and recombine with each other to give hybrid chromosomes which carry genes from both parent chromosomes. Meiosis is followed by the formation of spores which germinate to produce haploid mycelium.(BSc Microbiology Microbial Genetics Notes Study Material)

However, S. cerevisiae is non-mycelial. The diploid cells formed after fusion can undergo normal growth and division. There are no fruit bodies, and each diploid cell can undergo meiosis to produce haploid cells.

In eukaryotic microbes, there is alternation of a haploid and a diploid generation. In different forms, there is a wide variation in the relative importance of the haploid and diploid phases for vegetative reproduction and in the degree of sexual differentiation they exhibit. In fungi, the predominant phase may be haploid or diploid or there may be roughly equal role of both; there may be morphologically distinct male and female forms or there may be sexual conjugation between two similar cells derived from the same clone.

An important characteristic of eukaryotes is the presence of extra-nuclear DNA in organelles such as chloroplasts and mitochondria. Eukaryotic cells may also possess plasmids. S. cerevisiae has a 2µm plasmid that has formed the basis of gene cloning in yeast both in its own right and as a hybrid molecule with bacterial plasmids allowing the analysis of yeast genes both in bacteria and in the yeast itself.(BSc Microbiology Microbial Genetics Notes Study Material)

Genetic Engineering

Experimental work on bacterial recombination took a new dimension in the late 1970s when it became possible to insert genes into a bacterial DNA and thereby to establish a cell line that could produce proteins according to the instructions of microbiologists.

In fact the real interest in genetic manipulations increased in the 1960s when a group of bacterial enzymes called endonucleases was discovered. These enzymes cleave the phosphate-sugar bonds in the backbone of nucleic acids and could be used to open a bacterial chromosome at desired point. Moreover each endonuclease scanned a DNA chain and cleaved it at a restricted point. For this reason the endonucleases came to be known as restriction enzymes. The existence of these enzymes was first postulated by Werner Arber of the University of Basil (Switzerland), when he noted the bacterial enzymes scissoring viral DNA at selected spots. Hamilton Smith of Johns Hopkins University subsequently isolated a restriction enzyme from Gram-negative rod, Haemophilus influenzae. Daniel Nathans, of Johns Hopkins University, utilised the enzyme in 1971 to split the DNA of the monkey tumor virus, Simian virus₄₀ (SV₄₀). In 1978, the Nobel Prize was awarded to the three scientists. By that time over 100 restriction enzymes had been isolated and characterised. Among the first scientists to attempt a genetic manipulation was Paul Berg of Stanford University. In 1971, Berg and his coworkers opened the DNA molecule of SV₄₀ and spliced it to a bacterial chromosome. In doing so they constructed the first recombinant DNA molecule.(BSc Microbiology Microbial Genetics Notes Study Material)

However, the process was very tedious because the bacterial and viral DNAs had blunt ends. Berg used much chemistry to form staggered ends. He later shared the 1980 Nobel Prize in Chemistry with Frederick Sanger. While Berg was performing his experiments in 1971, an important breakthrough came from Herbert Boyer and co-workers at Univ. of California, San Francisco. U.S.A. Boyer isolated a restriction enzyme that would nick a chromosome cleanly and leave it with mortise-like staggered ends. The bits of single-stranded DNA extending out the chromosome easily attached to a new fragment of DNA in recombinant experiments. Scientists quickly dubbed the single-stranded extensions “sticky ends”. During the same period, Stanley Cohen at Stanford University was working with plasmids of E. coli. He found that he could isolate plasmids easily from the cell and then insert them into fresh bacteria by suspending the organisms in calcium chloride and heating them suddenly to achieve transformation. Once inside E.coli cells the plasmids multiplied independently and produced copies of themselves in succeeding generations. Cohen’s data thus indicated that it was now unnecessary to work with the larger, less-manageable chromosome. One may well work with plasmids.

The final link to the process was provided by the DNA ligases. These enzymes known since the 1960s function in the replication of DNA and the repair of broken DNA molecules. Essentially they operate in a manner opposite to the endonucleases, that is, they seal together DNA fragments. Working together Boyer and Cohen isolated plasmids from E. coli and opened them with restriction enzymes. Then they inserted a segment of foreign DNA using DNA ligase. Then they implanted the plasmids into fresh E. coli. By 1973, Boyer and Cohen had successfully spliced genes from Staphylococcus aureus into E. coli. The recombined plasmids were called chimeras. Paul Berg in 1973 proposed to splice SV₄₀ genes to E. coli using the techniques of Boyer and Cohen. Berg was using SV₄₀ virus because it contained only seven genes and was therefore easy to work with. Other scientists, learning of the proposal, reminded Berg that if the recombined E. coli escaped from the laboratory, it might settle in the human intestine and carry along the tumor genes of the SV₄₀ virus. Berg cancelled the idea, and with ten other scientists warned for the potential dangers of recombinant DNA technology. In 1974, scientists called a moratorium on all such experiments However, scientists since then review the consequences of such techniques. Only the most risky ones remain under regulation. In 1985, one such experiment, involving release of bacteria into environment stirred much controversy. Pseudomonas syringae is present in the phylloplane of many plants. At temperatures below 32°F it produces a protein that induces ice crystal formation thus causing frost injury to potato plants. Steven Lindow and Nickolas Panapoulas of the Univ. of California in 1983 enginerred the bacterium that now lacked the culprit protein. Plants were now able to withstand temperature as low as 23°F because dew can cool to that point before it turns to ice. Plants were protected from frost and their growing season was also extended. The spray was banned by court in 1984. However, permission was granted in 1985 by the court.

Gene cloning procedure

Genetic engineering rests on two of the major discoveries of the last 20 years, namely, plasmids and restriction enzymes. Plasmids have already been described. We have already indicated about restriction enzymes also. They are in fact one of the bacterial defense systems against entry of foreign DNA. The restriction enzyme recognises a specific sequence of DNA bases and cuts the DNA at or close to that site. This specific sequences of bases occurs within the DNA of the bacterium but is protected from the action of the restriction enzyme by modification of one of the bases within the sequence. Foreign DNA is not similarly protected so that when it enters the cell it is rapidly degraded by the restriction enzyme.(BSc Microbiology Microbial Genetics Notes Study Material)

The plasmids used in genetic engineering are relatives of the naturally occurring plasmids found in microorganisms. Generally they have been mutated and tailored with enzymes in order to have specific desired properties. Through genetic engineering (as an advantage over conventional genetic techniques) one can transfer a single specific gene between organisms. The steps are:

(1) The donor DNA carrying the gene of interest is cut with a restriction enzymeto yield fragments of various sizes, one of which bears the desired gene. A suitable plasmid cut with the same enzyme is mixed with the donor DNA and the two are joined by an enzyme, DNA ligase, to give a series of hybrid plasmids.

(2) The hybrid plasmid DNA is used to transform the host cell and the cells are plated out onto agar. The ratio of the amount of DNA to cells is adjusted in such a way that each cell takes up only one DNA molecule. Thus each colony which grows represents a different piece of the DNA donor carried on a plasmid. One of these carries the gene of interest and can be recognised by the characteristics it confers on the cell. This gene is then said to have been cloned. This is the basis of genetic engineering. This method can be applied to DNA from any organisms and the host can be a bacterium, yeast, other fungus or plant or animal cell. Genetic engineering has made significant contributions to industrial microbiology and in understanding the possible mechanisms of adaptability in microorganisms.

In brief gene cloning involves (i) isolation and fragmentation of the source DNA and incorporation of the fragments obtained into a cloning vector (segment of DNA used for replication of foreign DNA fragments), with the use of restriction endonucleases to cut and ligase to rejoin DNA molecules (ii) incorporation of the genetically transformed DNA into a recipient organism that can replicate the cloning vector, (iii) detection of the newly transformed cells containing DNA and isolation of a pure culture therefrom; and (iv) growth of culture of cells containing the cloned DNA fragment. A cloning vector must be able to replicate autonomously in a suitable host. Plasmids are used as carriers of unrelated DNA for genetic engineering, and the enzymes used for splicing foreign DNA into the plasmid carriers are those used in normal recombination and replication of DNA.

However, at the same time there are dangers also associated with this technology. There are possibilities of creating new and uncontrollable pathogens from tame microbes like E. coli and yeast.

Modern Applications of Genetic Engineering

Genetic engineering has wide applications in modern biotechnology. For various industrial processes, this technique may be used in microorganisms as well as with higher organisms. The principle involved is the construction of plasmids of desired biochemical characteristics. Plasmid technology is being hailed by many as the beginning of modern industrial microbiology. The plasmids are tiny ringlets of DNA, apart from the chromosome, that may contain 2-250 genes. They exist autonomously in the cell. The plasmids can be spliced with genes from an unrelated organism. The genes now function to produce the protein (of unrelated organism) in the cell of host microorganism. The following are the chief possible applications:

(1) Plasmids of one bacterium may be spliced with genes from other bacterium. For example it is found that plasmids of Pseudomonas will function in other Gram-negative bacteria as Escherichia, Proteus or Rhizobium, and that staphylococcal plasmids can be transferred to Bacillus subtilis cells, where they will replicate and express themselves.(BSc Microbiology Microbial Genetics Notes Study Material)

(2) Since microbial cells have a much higher metabolic rate, genes of desired enzymes (of commercial values) could be introduced into plasmid of bacteria. For instance genes of amylase synthesis could be derived from yeasts by introducing plasmid genes for amylase production. This would enhance the process of beer fermentations. Similarly genes for cellulase synthesis could be incorporated into plasmids of microbes. The resulting large scale cellulases could be utilised for cellulose degradation.(BSc Microbiology Microbial Genetics Notes Study Material)

(3) Even nitrogen fertilisers may be eliminated by incorporating plasmids, containing bacterial genes for nitrogen fixation, into the plant cells.

(4) Plasmid technology has shown that products like insulin, interferon, vaccines and human growth hormones may be industrially possible. By 1984, over 200 companies world over had established gene-splicing experiments, and working on industrial applications of genetic engineering. One company in 1980 could harvest insulin from bacteria whose plasmids had been spliced with DNA for this protein. The DNA was from chromosome number 11 of human cells, thus product was identical with human insulin. Marketed by Eli Lilly Corporation, the bacterial insulin, humulin, is identical with human insulin. In 1980, interferon was produced by genetic engineering from bacterial cells. By 1986, interferon from engineered bacteria was being tested on rabies victims, common cold patients and cancer patients. Scientists have also inserted genes into bacteria for the production of human growth hormone. This hormone is used to treat dwarfism. In 1986, the hormone became commercially available as protropin. In June 1981, a vaccine for foot-and-mouth disease was developed by genetic engineering firm. There are many other products derived from genetic engineering. Urokinase, a clot-dissolving enzyme is produced from genetically engineered bacteria. Endorphin, a pain-killer is also derived from bacteria. Bacteria have also been engineered to live solely on toxic wastes in the environment. A gene for hair-digesting enzyme is inserted into plasmid of bacteria.

There are also attempts to engineer plants with bacterial genes that trap N₂ and convert it to a form that could be easily taken by the plant. Yeasts are being engineered to yield enzymes for cheese industry.

BSc Microbiology Microbial Genetics Notes Study Material

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