CHAPTER ONE

1.0 Introduction

1.1 Background Study

Microbial genetics is a subject area within microbiology and genetic engineering. Microbial

genetics studies microorganisms for different purposes. The microorganisms that are

observed are bacteria, and archaea. Some fungi and protozoa are also subjects used to study

in this field. The studies of microorganisms involve studies of genotype and expression

system. Genotypes are the inherited compositions of an organism. (Austin, "Genotype," n.d.)

Genetic Engineering is a field of work and study within microbial genetics. The usage of

recombinant DNA technology is a process of this work. The process involves creating

recombinant DNA molecules through manipulating a DNA sequence. That DNA created is

then in contact with a host organism. Cloning is also an example of genetic engineering.

(microbes and the tools of genetic engineering).

Since the discovery of microorganisms by Robert Hooke and Antoni van Leeuwenhoek

during the period 1665-1885[Gest and hau] they have been used to study many processes and

have had applications in various areas of study in genetics. For example: Microorganisms'

rapid growth rates and short generation times are used by scientists to study evolution. Robert

Hooke and Antoni van Leeuwenhoek discoveries involved depictions, observations, and

descriptions of microorganisms. Mucor is the micro fungus that Hooke presented and gave a

depiction of. His contribution being, Mucor as the first microorganism to be illustrated.

Antoni van Leeuwenhoek’s contribution to the microscopic protozoa and microscopic

bacteria yielded to scientific observations and descriptions. These contributions were

accomplished by a simple microscope, which led to the understanding of microbes today and

continues to progress scientists understanding. Microbial genetics also has applications in

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being able to study processes and pathways that are similar to those found in humans such as

drug metabolism.

1.1.1 Applications of microbial genetics

Microbes are ideally suited for biochemical and genetics studies and have made huge

contributions to these fields of science such as the demonstration that DNA is the genetic

material, that the gene has a simple linear structure, that the genetic code is a triplet code, and

that gene expression is regulated by specific genetic processes. Jacques Monod and François

Jacob used Escherichia coli, a type of bacteria, in order to develop the operon model of gene

expression, which lay down the basis of gene expression and regulation. Furthermore, the

hereditary processes of single-celled eukaryotic microorganisms are similar to those in multi-

cellular organisms allowing researchers to gather information on this process as well.

Another bacterium which has greatly contributed to the field of genetics is Thermus

aquaticus, which is a bacterium that tolerates high temperatures. From this microbe scientists

isolated the enzyme Taq polymerase, which is now used in the powerful experimental

technique, Polymerase chain reaction(PCR). Additionally, the development of recombinant

DNA technology through the use of bacteria has led to the birth of modern genetic

engineering and biotechnology.

Using microbes, protocols were developed to insert genes into bacterial plasmids, taking

advantage of their fast reproduction, to make bio factories for the gene of interest. Such

genetically engineered bacteria can produce pharmaceuticals such as insulin, human growth

hormone, interferons and blood clotting factors. These bio factories are typically much

cheaper to operate and maintain than the alternative procedures of producing

pharmaceuticals. They're like millions of tiny pharmaceutical machines that only require

basic raw materials and the right environment to produce a large amount of product. The

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utilization of incorporating the human insulin gene alone has had profound impacts on the

medical industry. It is thought that bio factories might be the ultimate key in reducing the

price of expensive lifesaving pharmaceutical compounds.

Microbes synthesize a variety of enzymes for industrial applications, such as fermented

foods, laboratory test reagents, dairy products (such as renin), and even in clothing (such as

Trichoderma fungus whose enzyme is used to give jeans a stone washed appearance).

There is currently potential for microbes to be used as an alternative for petroleum-based

surfactants. Microbial surfactants would still have the same kind of hydrophilic and

hydrophobic functional groups as their petroleum-based counterparts, but they have

numerous advantages over their competition. In comparison, microbial amphiphilic

compounds have robust a tendency to stay functional in extreme environments such as areas

with high heat or extreme ph. all while being biodegradable and less toxic to the

environment. This efficient and cheap method of production could be the solution to the ever-

increasing global consumption of surfactants. Ironically, the application for bio-based

surfactants with the most demand is the oil industry which uses surfactants in general

production as well as development of specific oil compositions.

Microbes are an abundant source of lipases which have a wide variety of industrial and

consumer applications. Enzymes perform a wide variety of functions inside the cells of living

things, so it only makes sense that we can use them for similar purposes on a larger scale.

Microbial enzymes are typically preferred for mass production due to the wide variety of

functions available and their ability to be mass produced. Plant and animal enzymes are

typically too expensive to be mass-produced, however this is not always the case. Especially

in plants. Industrial applications of lipases generally include the enzyme as a more efficient

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and cost-effective catalyst in the production of commercially valuable chemicals from fats

and oils, because they are able to retain their specific properties in mild easy to maintain

conditions and work at an increased rate. Other already successful applications of lipolytic

enzymes include the production of biofuels, polymers, non-stereoisomeric pharmaceuticals,

agricultural compounds, and flavour-enhancing compounds.

In regards to industrial optimization, the benefit of the bio factory method of production is the

ability to direct optimization by means of directed evolution. The efficiency and specificity of

production will increase over time by imposing artificial selection. This method of improving

efficiency is nothing new in agriculture, but it's a relatively new concept in industrial

production. It is thought that this method will be far superior to conventional industrial

methods because you have optimization on multiple fronts. The first front being that the

microorganisms that make up bio factories can be evolved to our needs. The second front

being the conventional method of optimization brought about by the integration of advancing

technologies. This combination of conventional and biological advancement is just now

becoming utilized and provides a virtually limitless number of applications.

1.1.2 Brief history of microbial genetics

- Microbial genetics began with the discovery of the first microorganisms by Antoine van

Leeuwenhoek in 1676.

- In the late 19th century, Louis Pasteur and Robert Koch developed techniques for

culturing and studying microorganisms.

- The discovery of DNA structure by James Watson and Francis Crick in 1953

revolutionized the field.

Key Concepts

- Microbial cells: Prokaryotic (lacking a nucleus) and eukaryotic (with a nucleus)

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 - DNA replication and transcription

- Genetic variation: Mutation, gene transfer, and recombination

- Gene regulation: Operons, transcription factors, and gene expression

- Genetic engineering: Recombinant DNA technology and gene editing (CRISPR)

Microorganisms have unique genetic features, such as:

- Horizontal gene transfer (sharing genes with other microorganisms)

- High mutation rates

- Ability to form biofilms and survive extreme environments

Understanding microbial genetics has led to significant advances in fields like:

- Biotechnology (antibiotics, vaccines, and enzyme production)

- Medicine (understanding and combating microbial diseases)

- Agriculture (soil microbiology and crop improvement)

- Environmental science (bioremediation and ecological balance)

Microbial genetics is a field of study that focuses on microorganisms such as bacteria,

archaea, fungi and protozoa. The study of microorganisms involves the study of genotypes,

which are the inherited compositions of an organism. Genetic engineering, which involves

the use of recombinant DNA technology, is a field of work and study within microbial

genetics. The process involves creating recombinant DNA molecules through manipulating a

DNA sequence. This DNA is then introduced into a host organism. Cloning is also an

example of genetic engineering.

Microorganisms have been used to study many processes and have had applications in

various areas of study in genetics. For example, microorganisms' rapid growth rates and short

generation times are used by scientists to study evolution. Microbial genetics also has

applications in being able to study processes and pathways that are similar to those found in

humans, such as drug metabolism.

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Microbial genetics can focus on Charles Darwin's work, and scientists have continued to

study his work and theories by the use of microbes. Specifically, Darwin's theory of natural

selection is a source used. Studying evolution by using microbial genetics involves scientists

looking at evolutionary balance. An example of how they may accomplish this is studying

natural selection or drift of microbes.

Microorganisms whose study is encompassed by microbial genetics include bacteria, archaea,

fungi, and protozoa. Bacteria have been on this planet for approximately 3.5 billion years and

are classified by their shape. Bacterial genetics studies the mechanisms of their heritable

information, their chromosomes, plasmids, transposons, and phages. Archaea is a domain of

organisms that are prokaryotic, single-celled, and are thought to have developed 4 billion

years ago. Fungi can be both multicellular and unicellular organisms and are distinguished

from other microbes by the way they obtain nutrients. Protozoa are unicellular organisms,

which have nuclei, and ultramicroscopic cellular bodies within their cytoplasm

1.2 Microbial genetics

Let’s talk about sex. Bacterial sex. Ha! That is going to be difficult, since bacteria do not

have sex. Which presents a real problem for bacteria (and archaea, too) – how do they get the

genetic variability that they need? They might need a new gene to break down an unusual

nutrient source or degrade an antibiotic threatening to destroy them – acquiring the gene

could mean the difference between life and death. But where would these genes come from?

How would the bacteria get a hold of them? We are going to explore the processes that

bacteria use to acquire new genes, the mechanisms known as Horizontal Gene Transfer

(HGT).

Conjugation

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Conjugation is the process by which a donor bacterium transfers a copy of a plasmid to a

recipient bacterium, through a pilus. The process requires cell-to-cell contact. The donor cell

(F+) has a conjugative plasmid, an extrachromosomal piece of dsDNA that codes for the

proteins necessary to make a threadlike filament known as a pilus. The pilus is used to bind to

the recipient (F-) cell, bringing it in close proximity to the donor cell. It is believed that a

channel is then opened between the two cells, allowing for a ssDNA copy of the plasmid to

enter the recipient cells. Both cells then make the complementary copy to the ssDNA,

resulting in two F+ cells capable of conjugation.

Figure1.0 Conjugation

Transformation

The process of transformation also allows a bacterial cell to acquire new genes, but it does

not require cell-to-cell contact. In this process the new genes are acquired directly from the

environment. Typically, the process requires a donor cell that at some point lysed and

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released naked DNA to the environment. The recipient cell is one that is capable of taking up

the DNA from the environment and incorporating it into its own genome, where the cell is

described as being competent.

There are mechanical and chemical means of encouraging a cell to pick up DNA from the

environment, but natural competence is determined genetically. The process typically occurs

at the end of exponential phase of growth or beginning of the stationary phase, in the

presence of high cell density and limited nutrients. Under these conditions specific proteins

are manufactured including DNA-binding proteins (DNA translocase), endonucleases, and

transmembrane channel proteins. Gram negative cells also make a cell wall autolysin, to

transport the DNA across the outer membrane.

Random pieces of DNA bind to receptors on the outside of the cell and are then transported

into the cell by the DNA translocase, through the transmembrane channel, a large structure

often involving numerous different proteins. An endonuclease can be used to degrade one

strand of dsDNA, if only ssDNA may pass into the cell, or to cleave the DNA fragment into

smaller sizes. Once inside the cell, the DNA must be incorporated into the bacterial

chromosome by RecA (see Molecular Recombination below), for the genes to be expressed.

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Figure 1.1: Gene Acquisition via Transformation

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Transduction

Transduction involves the use of a virus, a bacteriophage, to act as a conduit for shuttling

bacteria genes from one cell to another, thus negating the necessity for cell-to-cell contact.

There are two different types of transduction: generalized transduction and specialized

transduction.

Generalized Transduction

In generalized transduction, a bacterial host cell is infected with either a virulent or a

temperate bacteriophage engaging in the lytic cycle of replication. After the first three steps

of replication (absorption, penetration, and synthesis), the virus enters into the assembly

stage, during which fully formed virions are made. During this stage, random pieces of

bacterial DNA are mistakenly packaged into a phage head, resulting in the production of a

transducing particle. While these particles are not capable of infecting a cell in the

conventional sense, they can bind to a new bacterial host cell and inject their DNA inside. If

the DNA (from the first bacterial host cell) is incorporated into the recipient’s chromosome,

the genes can be expressed.

Figure 1.2: Generalized Transduction.

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Specialized Transduction

Specialized transduction can only occur with temperate bacteriophage, since it involves the

lysogenic cycle of replication. The bacteriophage randomly attaches to a bacterial host cell,

injecting viral DNA inside. The DNA integrates into the chromosome of the host cell,

forming a prophage. At some point induction occurs, where the prophage is excised from the

bacterial chromosome. In specialized transduction, the excision is incorrectly performed and

a portion of bacterial genes immediately adjacent to the viral genes are excised too. Since this

DNA is used as the template for the synthesis stage, all copies will be a hybrid of viral and

bacterial DNA, and all resulting virions will contain both viral and bacterial DNA.

Once the cell is lysed, the virions are released to infect other bacterial host cells. Each virion

will attach to the host cell and inject in the DNA hybrid, which could be incorporated into the

host chromosome, if a prophage is formed. At this point the second bacterial host cell can

contain its own DNA, DNA from the previous bacterial host cell, and viral DNA.

Figure 1.3: Specialized transduction

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Molecular Recombination

In each of the cases of HGT, the process is only successful if the genes can be expressed by

the altered cell. In conjugation, the genes are located on a plasmid, under the control of

promoters on the plasmid. In transformation and transduction, where naked DNA is gaining

access to the cell, the DNA could easily be broken down by the cell with no genetic

expression occurring. In order for the genes to be expressed, the DNA must be recombined

with the recipient’s chromosome.

The most common mechanism of molecular recombination is homologous recombination,

involving the RecA protein. In this process DNA from two sources are paired, based on

similar nucleotide sequence in one area. An endonuclease nicks one strand, allowing RecA to

pair up bases from different strands, a process known as strand invasion. The cross-over

between DNA molecules is resolved with resolvase, which cuts and re-joins the DNA into

two separate dsDNA molecules.

Recombination can also occur using site-specific recombination, a process often used by

viruses to insert their genome into the chromosome of their host. This type of recombination

is also used by transposable elements (see next section).

Transposable Elements

Finally, we shouldn’t leave the topic of microbial genetics without at least exploring the role

of transposable elements or “jumping genes.” While these can play a very big role in the

activation and inactivation of bacterial genes, the best explanation derives from the work of

Barbara McClintock in corn, who won the Nobel Prize for her research in 1983. She

demonstrated that transposable elements can be responsible for the activation or inactivation

of genes within an organism.

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Transposable elements are relatively simple in structure, designed to move from one location

to another within a DNA molecule by a process known as transposition. All transposable

elements code for the enzyme transposase, the enzyme responsible allowing transposition to

occur, and have short inverted repeats (IRs) at each end.

Figure1.4: Transposition

Transposition.

The simplest transposable element is an insertion sequence (IS), which contains the

transposase and IRs of varying lengths. A transposon typically contains additional genes,

with the exact type varying widely from transposon to transposon. A transposon can be

removed from one location and relocated to another (the cut-and-paste model), a process

known as conservative transposition. Alternatively, it can be copied, with the copy being

inserted at a second site, in a process known as replicative transposition.

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 CHAPTER TWO

2.1 Genetic Variation and Mutation in microorganisms

This section covers the sources and types of genetic variation in microorganisms, including

1. Mutation: This is a spontaneous change in the genetics composition of the cells

2. Causes of mutation: which include point mutation, nonsense mutation, missense mutation,

silent mutation, frame shift and so on

3. Recombination: Shuffling of genetic material between homologous chromosomes or genes,

increasing genetic diversity.

Understanding genetic variation and mutation is crucial for microbial genetics research and

applications. (Albert, B., Johnson, A., Lewis, J., Raff, M., Roberts, W., Walter, P.,2015).

2.2 Types of microbial mutations

1. Point mutations: Changes in a single nucleotide base in the DNA sequence. (Kouzminova,

2017)

2. Frameshift mutations: Insertions or deletions of one or more nucleotides, leading to

changes in the reading frame of the genetic code. (Kouzminova, 2017)

3. Gene duplication: Copies of a gene or genetic region, which can lead to increased gene

expression or the evolution of new functions. (Zhang, 2019)

4. Chromosomal mutations: Changes in the number or structure of chromosomes, such as

deletions, duplications, or translocations. (Liu, 2018)

5.Spontaneous mutations: Occur naturally, without any external influence, due to errors

during DNA replication or repair. (Chen, 2020)

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2.3 Causes of mutation

1.DNA spontaneously breaks down or is not copied accurately:

Most of the mutations that we think matter to evolution are “naturally-occurring.” For

example, when a cell divides, it makes a copy of its DNA — and sometimes the copy is not

quite perfect. That small difference from the original DNA sequence is a mutation.

Spontaneous breakdown of DNA can also cause mutations.

2. External influence can cause mutations:

Mutations can also be caused by exposure to specific chemicals or radiation that cause the

DNA to break down. Cells do have mechanisms to repair damaged or altered DNA

molecules, but they aren’t perfect. Whatever the cause, mutations occur any time a cell ends

up carrying a DNA sequence slightly different than the original. (Wu, F., Strand, A., Cox, L.,

Ober, C., Wall, J., Moorjani, P., and Przeworski, M. 2020).

In humans, each baby has around 70 brand new or “de novo” mutations. De novo mutations

occur in the reproductive cells of parents and are passed on to the child. Evidence suggests

that most de novo mutations in a child come from the sperm that helped create that child, and

relatively few mutations come from the egg. Biologists thought this made sense. In humans,

beginning at puberty, the cells that produce sperm divide (and copy their DNA) throughout

adulthood, leading to vast numbers of sperm. In contrast, in a person with ovaries, all the

DNA copying leading up to egg production is completed before that person is even born.

(Wu, F.,2020)

Types of mutagenic agents include:

1. Physical mutagens:

A. Ionizing radiations (X-rays and gamma rays)

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B. Non-ionizing radiations (Ultraviolet and visible light)

C. Temperature increase

2. Chemical mutagens:

A. Base analogs (5-Bromouracil and aminopurine)

B. Intercalating agents (Ethidium bromide and proflavine)

C. Metal ions (Nickel, Chromium, and Cobalt)

D. Alkylating agents (Nitrous acid and ethyl nitrosourea)

3. Biological mutagens:

A. Transposons and Insertion sequences (IS)

B. Viruses (Rous sarcoma virus)

C. Bacteria (Helicobacter pylori)

2.4 Recombination

Recombination is a key mechanism in genetics that involves the exchange of genetic material

between different organisms, leading to offspring with new combinations of traits. Here are

some points about recombination and its types

- Genetic recombination occurs naturally during meiosis, when maternal and paternal genes

are regrouped to form gametes (sex cells).

- Crossing over occurs when gene sequences called linkage groups are disrupted, resulting in

an exchange of segments between paired chromosomes.

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- There are two main types of recombination: interchromosomal (occurring through

independent assortment of alleles on different but homologous chromosomes) and

intrachromosomal (occurring through crossing over).

- Recombination can also occur during mitosis, but it is less common and usually involves the

two sister chromosomes formed after chromosomal replication.

- Recombination is catalysed by enzymes called recombinases, which facilitate the strand

transfer step during recombination.

- Chromosomal crossover is the process by which recombination occurs, leading to offspring

having different combinations of genes from those of their parents.

- Gene conversion is the process by which a DNA sequence is copied from one chromosome

to another without changing the donating chromosome.

- Nonhomologous recombination can occur between DNA sequences with no sequence

homology and can cause chromosomal translocations, leading to cancer. (Lodish, H., Berk,

A., & Kaiser, C. A. ,2020).

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 CHAPTER THREE

3.1 DNA replication processes in prokaryotes and eukaryotes

DNA replication is semiconservative. Each strand in the double helix acts as a template for

synthesis of a new, complementary strand.

New DNA is made by enzymes called DNA polymerases, which require a template and a

primer (starter) and synthesize DNA in the 5’ to 3’ direction.

During DNA replication, one new strand (the leading strand) is made as a continuous piece.

The other (the lagging strand) is made in small pieces.

DNA replication requires other enzymes in addition to DNA polymerase, including DNA

primase, DNA helicase, DNA ligase, and topoisomerase.

3.2 DNA replication

The elucidation of the structure of the double helix by James Watson and Francis Crick in

1953 provided a hint as to how DNA is copied during the process of replication. Separating

the strands of the double helix would provide two templates for the synthesis of new

complementary strands, but exactly how new DNA molecules were constructed was still

unclear. In one model, semiconservative replication, the two strands of the double helix

separate during DNA replication, and each strand serves as a template from which the new

complementary strand is copied; after replication, each double-stranded DNA includes one

parental or “old” strand and one “new” strand. There were two competing models also

suggested: conservative and dispersive

There were three models suggested for DNA replication. In the conservative model, parental

DNA strands (blue) remained associated in one DNA molecule while new daughter strands

(red) remained associated in newly formed DNA molecules. In the semiconservative model,

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parental strands separated and directed the synthesis of a daughter strand, with each resulting

DNA molecule being a hybrid of a parental strand and a daughter strand. In the dispersive

model, all resulting DNA strands have regions of double-stranded parental DNA and regions

of double-stranded daughter DNA.

Figure 2.0: DNA replication

3.3 Steps in DNA replication

Initiation

The initiation of replication occurs at specific nucleotide sequence called the origin of

replication, where various proteins bind to begin the replication process. E. coli has a single

origin of replication (as do most prokaryotes), called oriC, on its one chromosome. The origin

of replication is approximately 245 base pairs long and is rich in adenine-thymine (AT)

sequences.

Some of the proteins that bind to the origin of replication are important in making single-

stranded regions of DNA accessible for replication. Chromosomal DNA is typically wrapped

around histones (in eukaryotes and archaea) or histone-like proteins (in bacteria), and is

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supercoiled, or extensively wrapped and twisted on itself. This packaging makes the

information in the DNA molecule inaccessible. However, enzymes called topoisomerases

change the shape and supercoiling of the chromosome. For bacterial DNA replication to

begin, the supercoiled chromosome is relaxed by topoisomerase II, also called DNA gyrase.

An enzyme called helicase then separates the DNA strands by breaking the hydrogen bonds

between the nitrogenous base pairs. Recall that AT sequences have fewer hydrogen bonds

and, hence, have weaker interactions than guanine-cytosine (GC) sequences. These enzymes

require ATP hydrolysis. As the DNA opens up, Y-shaped structures called replication forks

are formed. Two replication forks are formed at the origin of replication, allowing for

bidirectional replication and formation of a structure that looks like a bubble when viewed

with a transmission electron microscope; as a result, this structure is called a replication

bubble. The DNA near each replication fork is coated with single-stranded binding proteins

to prevent the single-stranded DNA from rewinding into a double helix.

Once single-stranded DNA is accessible at the origin of replication, DNA replication can

begin. However, DNA pol III is able to add nucleotides only in the 5’ to 3’ direction (a new

DNA strand can be only extended in this direction). This is because DNA polymerase

requires a free 3’-OH group to which it can add nucleotides by forming a covalent

phosphodiester bond between the 3’-OH end and the 5’ phosphate of the next nucleotide.

This also means that it cannot add nucleotides if a free 3’-OH group is not available, which is

the case for a single strand of DNA. The problem is solved with the help of an RNA sequence

that provides the free 3’-OH end. Because this sequence allows the start of DNA synthesis, it

is appropriately called the primer. The primer is five to 10 nucleotides long and

complementary to the parental or template DNA. It is synthesized by RNA primase, which is

an RNA polymerase. Unlike DNA polymerases, RNA polymerases do not need a free 3’-OH

group to synthesize an RNA molecule. Now that the primer provides the free 3’-OH group,

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DNA polymerase III can now extend this RNA primer, adding DNA nucleotides one by one

that are complementary to the template strand.

Elongation

During elongation in DNA replication, the addition of nucleotides occurs at its maximal rate

of about 1000 nucleotides per second. DNA polymerase III can only extend in the 5’ to 3’

direction, which poses a problem at the replication fork. The DNA double helix is

antiparallel; that is, one strand is oriented in the 5’ to 3’ direction and the other is oriented in

the 3’ to 5’ direction. During replication, one strand, which is complementary to the 3’ to 5’

parental DNA strand, is synthesized continuously toward the replication fork because

polymerase can add nucleotides in this direction. This continuously synthesized strand is

known as the leading strand. The other strand, complementary to the 5’ to 3’ parental DNA,

grows away from the replication fork, so the polymerase must move back toward the

replication fork to begin adding bases to a new primer, again in the direction away from the

replication fork. It does so until it bumps into the previously synthesized strand and then it

moves back again (Figure 1.6). These steps produce small DNA sequence fragments known

as Okazaki fragments, each separated by RNA primer. Okazaki fragments are named after the

Japanese research team and married couple Reiji and Tsuneko Okazaki, who first discovered

them in 1966. The strand with the Okazaki fragments is known as the lagging strand, and its

synthesis is said to be discontinuous.

The leading strand can be extended from one primer alone, whereas the lagging strand needs

a new primer for each of the short Okazaki fragments. The overall direction of the lagging

strand will be 3’ to 5’, and that of the leading strand 5’ to 3’. A protein called the sliding

clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding

clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place.

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Beyond its role in initiation, topoisomerase also prevents the over winding of the DNA

double helix ahead of the replication fork as the DNA is opening up; it does so by causing

temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA

primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA

polymerase I, and the gaps are filled in. The nicks that remain between the newly synthesized

DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the

enzyme DNA ligase that catalyses the formation of covalent phosphodiester linkage between

the 3’-OH end of one DNA fragment and the 5’ phosphate end of the other fragment,

stabilizing the sugar-phosphate backbone of the DNA molecule.

Diagram of DNA replication. A small inset at the top shows a double strand of DNA

separated in the centre forming a bubble; the DNA is double stranded on either side of the

bubble. The origin of replication is in the midway point of the bubble. On the top strand a

solid arrow points to the left from the origin; this is the leading strand. On the right of the

origin of replication are short arrows pointing to the left; this is the lagging strand. On the

bottom strand a solid arrow pointing to the right from the origin is labelled leading strand and

short arrows pointing to the right on the other side of the origin are labelled lagging strands.

A larger image shows just the left half of the bubble. The double stranded DNA is no the far

left and is labelled 5’ for the top strand and 3’ for the bottom strand. An enzyme to the very

far left I is labelled topoisomerase/gyrase. At the point where the double stranded regions

splits are a triangle shape labelled helicase. Next to that are smaller shapes labelled single-

stranded binding proteins. The top strand shows continuous synthesis of the leading strand;

this is shown as a solid arrow under the top strand. The arrow has a 5’ at the right end and a

3’ at the left end. The template strand at the top has a 3’ at the right and a 5’ at the left. At the

end of the arrow (near where the DNA is newly being separated by the helicase) is DNA

polymerase 3 and a sliding clamp that span both strands. The bottom strand of DNA has more

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components. Just after the single stranded binding proteins is RNA primase which attaches

RNA primer (shown as a green arrow). Further down the lagging strand template is an

existing RNA primer with DNA polymerase III and a sliding clamp spanning primer and the

template strand. The polymerase is building a new strand of DNA from the left side (5’) to

the right side (3’). Further to the right is a long piece made of RNA primer, then new DNA,

then RNA primer, then new DNA all connected. Each of the DNA/RNA combinations are

okazaki fragments made in the discontinuous synthesis of the lagging strand. DNA

polymerase I is attached to the RNA primer in the centre and is replacing it with DNA

nucleotides. DNA ligase then binds the individual strands of new DNA together. This is

shown in a close-up as two double helices that have all the correct letters in place, but one is

missing a connection between two of the nucleotides (this is called a single-stranded gap).

DNA ligase forms this last bond and the gap is sealed.

Figure 1.7 At the origin of replication, topoisomerase II relaxes the supercoiled chromosome.

Two replication forks are formed by the opening of the double-stranded DNA at the origin,

and helicase separates the DNA strands, which are coated by single-stranded binding proteins

to keep the strands separated. DNA replication occurs in both directions. An RNA primer

complementary to the parental strand is synthesized by RNA primase and is elongated by

DNA polymerase III through the addition of nucleotides to the 3’-OH end. On the leading

strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized

in short stretches called Okazaki fragments. RNA primers within the lagging strand are

removed by the exonuclease activity of DNA polymerase I, and the Okazaki fragments are

joined by DNA ligase.

Termination

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Once the complete chromosome has been replicated, termination of DNA replication must

occur. Although much is known about initiation of replication, less is known about the

termination process. Following replication, the resulting complete circular genomes of

prokaryotes are concatenated, meaning that the circular DNA chromosomes are interlocked

and must be separated from each other. This is accomplished through the activity of bacterial

topoisomerase IV, which introduces double-stranded breaks into DNA molecules, allowing

them to separate from each other; the enzyme then reseals the circular chromosomes. The

resolution of concatemers is an issue unique to prokaryotic DNA replication because of their

circular chromosomes. Because both bacterial DNA gyrase and topoisomerase IV are distinct

from their eukaryotic counterparts, these enzymes serve as targets for a class of antimicrobial

drugs called quinolones.

Figure 2.1 DNA replication showing the enzymes involved

At the origin of replication, topoisomerase II relaxes the supercoiled chromosome. Two

replication forks are formed by the opening of the double-stranded DNA at the origin, and

helicase separates the DNA strands, which are coated by single-stranded binding proteins to

25
keep the strands separated. DNA replication occurs in both directions. An RNA primer

complementary to the parental strand is synthesized by RNA primase and is elongated by

DNA polymerase III through the addition of nucleotides to the 3’-OH end. On the leading

strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized

in short stretches called Okazaki fragments. RNA primers within the lagging strand are

removed by the exonuclease activity of DNA polymerase I, and the Okazaki fragments are

joined by DNA ligase.

3.4. Key differences between prokaryotic and eukaryotic DNA replication

Eukaryotes usually have multiple linear chromosomes, each with multiple origins of

replication.

Most of the E. coli enzymes have counterparts in eukaryotic DNA replication, but a single

enzyme in E. coli may be represented by multiple enzymes in eukaryotes.

Most eukaryotic chromosomes are linear. Because of the way the lagging strand is made,

some DNA is lost from the ends of linear chromosomes (telomeres) in each round of

replication. (Toppr, 2020).

3.4.1. Comparison of Bacterial and Eukaryotic Replication

Property Bacteria Eukaryotes

Genome structure Singular circular Multiple linear
chromosome chromosomes
Number of origins per Single Multiple
chromosomes
Rate of replication 1000 nucleotides per second 100 nucleotides per second
Telomerase Not present Present
RNA primer removal DNA pol 1 Rnase H
Strand elongation DNA pol III Pol δ, pol ε

3.4.2. Mechanisms of DNA Replication Initiation and Elongation

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Mechanism of DNA replication initiation and elongation are as follows

1. Initiation:

- The double-stranded helical structure of DNA opens up, and the strands separate at a

specific site known as the origin.

- Several enzymes and proteins prepare the strands for duplication, including the binding of

the DnaA protein to the oriC locus in E. coli.

- Primers are added to the template strands.

2. Elongation:

- DNA polymerase enzyme starts assembling the new DNA strands.

- The leading strand is continuously synthesized in the 5’ to 3’ direction.

- The lagging strand is synthesized in short, discontinuous segments called Okazaki

fragments, which are later joined together by DNA ligase.

- RNA primers are removed, and the gaps are filled with DNA.

Note that the specific enzymes and proteins involved may vary slightly between prokaryotic

and eukaryotic cells. (Biology Libre Texts, 2024)

3.4.3. DNA Repair Mechanisms

There are several types of DNA repair mechanisms

- Direct reversal repair: This mechanism fixes specific types of DNA damage without the

need for excision or replacement.

- Base excision repair (BER): This mechanism removes and replaces damaged bases.

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- Nucleotide excision repair (NER): This mechanism deals with bulky adducts and cross-

linking lesions caused by UV radiation or chemical exposure.

- Mismatch repair (MMR): This mechanism repairs base mismatches and insertion-deletion

loops that occur during replication.

- Single-strand break repair (SSBR): This mechanism repairs single-stranded breaks in DNA.

- Double-strand break repair: This mechanism repairs double-stranded breaks in DNA

through two pathways: homologous recombination (HR) and non-homologous end joining

(NHEJ).

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 CONCLUSION

The study of microbial genetics has revealed the intricate mechanisms that govern the

structure, replication, and repair of microbial DNA. The double helix structure of DNA,

comprising nucleotides with sugar-phosphate backbones and nitrogenous bases, provides the

blueprint for microbial life. The processes of DNA replication, initiated by unwinding the

double helix and synthesizing new strands, ensure genetic continuity. Meanwhile, DNA

repair mechanisms, such as mismatch repair and homologous recombination, maintain

genomic integrity by correcting errors and damages.

The steps In DNA replication, including initiation, elongation, and termination, are crucial for

microbial growth and survival. Moreover, genetic variability, arising from mutations and

recombination, drives evolution and adaptation in microorganisms.

Understanding these fundamental processes has significant implications for various fields,

including:

- Development of antimicrobial therapies targeting DNA replication and repair

- Design of genetic engineering strategies for microbial biotechnology

- Elucidation of microbial evolution and adaptation mechanisms

- Development of novel DNA-based diagnostic tools

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 RECOMMENDATION

Further research is needed to fully understand the genetic mechanisms underlying microbial

evolution and adaptation, the development of novel genetic engineering tools and techniques

will revolutionize the field of microbial biotechnology, investigation into the genetic basis of

microbial symbiosis and pathogenesis will lead to a better understanding of microbial

interactions with their environments, the application of microbial genetics in bioremediation

and biodegradation has the potential to revolutionize environmental sustainability, genetic

manipulation of microbes for biofuel production and other industrial applications holds great

promise, elucidating the genetic mechanisms of antibiotic resistance will inform the

development of novel antimicrobial therapies, the study of microbial epigenetics and

epigenomics will reveal new insights into gene regulation and chromatin modification,

exploring the human microbiome will uncover the role of microbes in human health and

disease, development of DNA-based diagnostic tools will enable rapid detection and

identification of microorganisms, investigating the genetic basis of microbial stress responses

will inform the development of novel biotechnological applications.

30
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