Environmental Chemistry and Microbiology (WAS 713)

Topic: Classification of Microorganism

Introduction

Microorganisms are a diverse group of living organisms that include bacteria, viruses, fungi,
protozoa, and some microscopic algae. Appropriate classification is important for understanding
their biological ecological and clinical importance.

CLASSIFICATION OF LIFE

Microorganism are class in three main domine of life.

Bacterial

The Three Domains of Life

Microorganisms can be classified into three main domains based on their fundamental cellular
characteristics:
1. Archaea
 Single-celled microorganisms with unique cellular structures and biochemical
processes.
2. Found in extreme environments like hot springs, deep-sea vents, and hypersaline lakes.
e,g Chlorella (a unicellular green alga), Desmids, stonewort.

3. Bacteria
 The most abundant and diverse group of microorganisms.
 Typically have a simple cellular structure with a cell wall and cell membrane.
 Include both beneficial and pathogenic species.
examples pseudomonas putida, Nitrosomonas, Vibro cholerae
4. Eukarya
 Includes multicellular organisms like animals, plants, and fungi, as well as some
unicellular microbes.
 Characterized by membrane-bound organelles and a true nucleus.
 Relevant microorganisms include:
  Fungi - Eukaryotic microbes like yeasts and molds
 Protozoa - Single-celled eukaryotic microbes like amoebas and ciliates
 Microalgae - Microscopic photosynthetic eukaryotes

Bacterial Classification
Bacteria are further classified based on various morphological, physiological, and genetic
characteristics:
Gram Stain:
These classified bacteria into to broad groups Gram-positive vs Gram-negative, based on cell
wall composition
 Gram +ve: Staphylococcus aureus, Streptococcus pyrogenes, Clostridium tetan, Bacillus
anthracis Clostridium perfringes
 Gram -ve: Neisseria gonorrhoeae, Escherichia coli., Salmonella typhi.
Cell Shape:
Bacteria are classified into five groups based on their shapes
 Cocci are round-shaped bacteria; e.g. Staphylococcus aureus which cause skin infections.
 Bacilli are rod-shaped bacteria; e.g. Escherichia coli which cause diarrhea.
 Spirilli are spiral-shaped bacteria; e.g. Helicobacter pylori which cause gastric ulcers.
 Vibrios are comma-shaped bacteria; e.g. Vibrio cholerae which cause cholera.
 Spirochetes are corkscrew-shaped bacteria; e.g. Treponema pallidum which cause
syphilis.
Oxygen Requirements:
 Obligate Aerobes: Produce ATP via aerobic respiration. Require around 20% to 21
atmospheric oxygen. Eg., Bacillus spp., pseudomonas, Micrococcus Staphylococcus
spp..

 Microaerophiles (Microaerophilic bacteria): Produce ATP via aerobic respiration or

fermentation. Require between 2-15% atmospheric oxygen for growth. They cannot
tolerate the atmospheric air but require oxygen at a low level for growth. E.g.,
Treponema pallidum, Streptococcus pneumoniae, Helicobacter pylori, Campylobacter,
Streptococcus pyogenes, and Borrelia burgdorferi.
  Aerotolerant Anaerobes: Produce ATP via anaerobic respiration and can conduct
fermentation. Oxygen can be present, but they do not utilize it for ATP production or
fermentation. E.g., Lactobacillus, Streptococcus pyogenes, etc.
 Facultative Anaerobes: Produce ATP via aerobic respiration, anaerobic respiration,
and/or fermentation. These organisms grow equally well in aerobic or anaerobic
environments. E.g., E. coli, Klebsiella, Salmonella, Shigella, Proteus, Vibrio cholerae
etc.
 Obligate Anaerobes: Produce ATP via anaerobic respiration or fermentation. These
bacteria die in the presence of O 2 because they lack the enzymes needed to break down
toxic forms of oxygen and their intermediate byproducts. E.g.: Gram-positive
(Clostridium, Propionibacterium, Bifidobacterium), Gram-negative: (Bacteroids,
Fusobacterium)

Spore Formation:
This a survival technique used by bacteria in unfavorable condition, bacteria are classified into
 Spore-Forming: Eg Bacillus spp.
 Non-spore-forming Eg., E. coli, Staphylococcus aureus
Motility: Motile (with flagella) vs non-motile
Nutritional Requirements: autotrophic vs heterotrophic
 Autotrophic Bacteria (autotrophs): Bacteria can make their organic compounds
themselves. Its principal source of carbon utilization is CO2. E.g., Purple and green sulfur
bacteria
 Heterotrophic Bacteria (heterotrophs): Bacteria depend on the others performed organic
compounds. E.g, Escherichia coli, Salmonella Typhi, Proteus spp., Staphylococcus
aureus, Lactobacillus acidophilus, Azospirillum spp. etc.
Genetic Analysis: 16S rRNA gene sequencing, DNA-DNA hybridization
Temperature: Bacteria are also classified based on temperature.

Viral Classification
Viruses are classified based on their genetic material, capsid structure, and mode of replication:
 Genetic Material: DNA viruses (e.g. herpes viruses) vs RNA viruses (e.g. influenza
viruses)
 Capsid Symmetry: icosahedral, helical, complex
 Envelope: enveloped vs non-enveloped
 Replication Strategy: lytic vs lysogenic
Fungal and Protozoan Classification
 Fungi are classified based on spore production, cell wall composition, and reproductive
structures.
 Protozoa are classified based on mode of locomotion (e.g. flagella, pseudopodia, cilia)
and life cycle stages.
Understanding the classification of microorganisms is fundamental to studying their biology,
ecology, and clinical significance in the field of medical microbiology.
Lesson 4
PRESERVATION OF MICROOGANISMS
The preservation of microorganisms involves the act of deliberately keeping microorganisms
alive and in stable condition after isolation and identification for future use.
Preservation of microbial culture is imperative because it helps to maintain the viability and
genetic stability of microorganisms over time.

Technique in Microbial Preservation

There are different ways to preserve microorganisms which include;
1. Periodic Transfer to Fresh Media
Strains can be maintained by periodically preparing a fresh culture from the previous
stock. The culture medium, the storage temperature, and the time interval at which the
transfers are made vary with the species and must be ascertained beforehand. The
temperature and the medium chosen should support a slow rather than a rapid rate of
growth so that the time interval between transfers can be as long as possible. Many more
common heterotrophs remain viable for several weeks or months on a medium like
nutrient agar.
2. Preparation Slant
3. Freezing:
This technique involves putting the microorganisms in a special solution to prevent
damage during freezing. The storage is at very low temperatures (-80°C or lower) to
maintain their viability. Freezing is common for preserving bacteria and fungi. The
cryoprotective solution used in freezing typically contains a combination of salts, sugars,
and organic compounds that protect the cells during freezing. Researchers commonly use
glycerol, dimethyl sulfoxide (DMSO), and trehalose as cryoprotectants. After freezing,
researchers store the samples in liquid nitrogen (-196°C) or a deep freezer (-80°C) for
long-term preservation.
4. Refrigeration
Pure cultures can be successfully stored at 0-8°C in refrigerators or cold rooms. This
method can only preserved organism for a short duration (2-3 weeks for bacteria and 3-4
months for fungi) because the metabolic activities of the microorganisms are significantly
 slowed down but not stopped. Thus, their growth continues slowly, nutrients are utilized,
and waste products are released into the medium. This finally results in the microbes’
death after some time.
5. Freeze-drying or Lyophilization
Both freeze drying and lyophilization are methods for preserving microbes for long-term
storage. Researchers grow microbes in a culture medium, harvest them, and suspend
them in a solution with a cryoprotectant. Next the solution with microbe is frozen, then
dried in a vacuum chamber, and finally, a special agent is added to prevent damage from
freezing. The dry powder or pellet can be stored at room temperature for a long time.
Freeze-drying is useful for preserving bacterial cultures, yeast, and some types of viruses.
6. Mineral oil overlay
This technique is common for preserving bacterial cultures in liquid media. This method
is useful for short-term storage of bacterial and fungal cultures. This technique involves
covering the surface of a culture with mineral oil to prevent evaporation and
contamination. The mineral oil forms a layer on top of the culture, preventing air from
coming into contact with the cells and reducing the risk of contamination. Mineral oil
overlay can also help to maintain the pH of the medium.
7. Glycerol stocks
Glycerol stocks are useful for preserving bacterial and fungal cultures for many years.
This technique involves mixing the microorganisms with a glycerol solution and storing
the samples in a freezer for long-term preservation. Glycerol mainly prevents ice crystal
formation during freezing that can damage the cells. The concentration of glycerol used
in the solution can vary depending on the microorganism and the intended use of the
preserved sample. Typically, concentrations of 10-20% glycerol are used for bacterial
cultures, while concentrations of up to 50% are used for fungal cultures. After mixing the
cells with glycerol, the samples are usually stored at -80°C or in liquid nitrogen for long-
term preservation. The samples can be revived by thawing and plating on appropriate
growth media.
Benefits of microbial preservation

 Keeping microorganisms alive and stable for future use

 Ensuring that important traits and characteristics are maintained
 Providing a steady supply of microbial products
 Preventing the loss of rare or valuable microorganisms
 Study microorganisms over time
 Saving time and money on acquiring and cultivating microorganisms
Lesson 5 & 6
Microbial Genetics

RNA and DNA Replication

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are perhaps the most important
molecules in cell biology, responsible for the storage and reading of genetic information that
ensure continuity all life. They are both linear polymers, consisting of sugars, phosphates and
nitrogenous bases,

How are information transferred from one generation to another

There are two classes of genetic materials that are responsible for the transfer of information
from one generation to another in animals:

DNA or deoxyribonucleic acid

RNA or ribonucleic acid

It is in the DNA or RNA sequences that biological information is stored and passed on.

DNA

Most organisms contain DNA except some viruses which contain RNA as their genetic
material. DNA was discovered by two scientists- Watson and Crick and their model of the
structure of DNA are called the Watson and Crick model. DNA is in a double helix structure
made up of nucleotides. The "backbone" of the double helix is composed of phosphates
connected to a five-carbon sugar called deoxyribose. DNA molecule is a double helix
consisting of two strands. Each strand of this helix is made up of nucleotides. Each
nucleotide is made up of a phosphoric acid, a deoxyribose sugar and a nitrogenous base.
Nitrogenous bases are of two types, viz. purines and pyrimidines.

The purines are of two types, viz. adenine and guanine

Pyrimidines are of two types viz. cytosine and thymine.

The adenine always pairs with thymine with double hydrogen bonds

Cytosine always pairs with guanine with triple hydrogen bond.

 The helices remain bound due to these hydrogen bonds.

RNA:

Unlike the DNA, RNA is a single-stranded genetic material. The nucleotide bases present in
RNA are similar to those in DNA except that thymine is replaced by uracil and pairs with
adenine. While DNA is the genetic material in most organisms, RNA is found in a few viruses.
RNA is of three types depending on their function:

tRNA or transfer RNA- helps transfer the amino acids from the mRNA to the ribosomes.

mRNA or messenger RNA- helps to carry the codes for amino acids from the DNA to the
ribosomes

rRNA or ribosomal RNA- are found on the ribosomes and help in protein synthesis.

rRNA : It is the component of the ribosome. It helps in protein synthesis.

tRNA is present in the cytoplasm. According to the message of the mRNA, it carries the specific
amino acid up to the ribosomes as per the message coded on mRNA.

Transcription, Translation and Translocation:

(i) Transcription: Synthesis of mRNA as per the nucleotide sequence present on the DNA
molecule, is called the process of transcription.

 The nucleotide sequence present in the DNA molecule is called gene. Genes control the
structure and functioning of cells of the body.

 Information required for the synthesis of proteins is stored in the genes i.e. in the
nucleotide sequences of DNA. The proteins are synthesized according to the need of the
body.

 Central Dogma: Synthesis of proteins by DNA through the RNA is called central dogma.

 DNA Transcription →RNA Translation→ Protein

1. Transcription:

 Definition: Transcription is the process by which genetic information encoded in

DNA is used to synthesize RNA molecules, specifically messenger RNA
(mRNA).

 Process: It occurs in the cell nucleus, where the DNA serves as a template for the
synthesis of complementary mRNA strands. The enzyme RNA polymerase
catalyzes the formation of mRNA by matching complementary RNA nucleotides
to the DNA template.

2. Translation:

 Definition: Translation is the process in which the information carried by mRNA

is used to build a corresponding protein.

3. Translocation:

 Definition: Translocation has different meanings depending on the biological

context. In the context of genetics, translocation refers to the movement of a
chromosomal segment from one location to another.

 Genetic Translocation: This can occur between non-homologous chromosomes

or within the same chromosome. It can lead to genetic disorders or contribute to
genetic diversity.
  Cellular Translocation: In cellular biology, translocation can also refer to the
movement of molecules or cellular structures from one location to another within
a cell. For example, proteins may be translocated from the cytoplasm to specific
organelles or from the endoplasmic reticulum to the Golgi apparatus.

In summary, transcription involves the synthesis of mRNA from DNA, translation

involves the synthesis of proteins from mRNA, and translocation can refer to the
movement of genetic material within chromosomes or the movement of molecules within
a cell. Each process plays a crucial role in the central dogma of molecular biology, which
describes the flow of genetic information from DNA to RNA to protein.

Differences and Comparison Between RNA and DNA

A comparison of the helix and base structure of RNA and DNA. Credit: Technology Networks
Comparison DNA RNA

Full Name Deoxyribonucleic Acid Ribonucleic Acid

RNA converts the genetic information

DNA replicates and stores genetic
Function contained within DNA to a format used
information. It is a blueprint for all genetic
to build proteins, and then moves it to
information contained within an organism.
ribosomal protein factories.

RNA only has one strand, but like

DNA consists of two strands, arranged in a
DNA, is made up of nucleotides. RNA
double helix. These strands are made up of
Structure strands are shorter than DNA strands.
subunits called nucleotides. Each nucleotide
RNA sometimes forms a secondary
contains a phosphate, a 5-carbon sugar
double helix structure, but only
molecule and a nitrogenous base.
intermittently.

DNA is a much longer polymer than RNA. A RNA molecules are variable in length,
chromosome, for example, is a single, long but much shorter than long DNA
Length
DNA molecule, which would be several polymers. A large RNA molecule
centimetres in length when unraveled. might only be a few thousand base
pairs long.

The sugar in DNA is deoxyribose, which RNA contains ribose sugar molecules,
contains one less hydroxyl group than without the hydroxyl modifications of
Sugar
RNA’s ribose. deoxyribose.

RNA shares Adenine (‘A’), Guanine

The bases in DNA are Adenine (‘A’),
(‘G’) and Cytosine (‘C’) with DNA,
Bases Thymine (‘T’), Guanine (‘G’) and Cytosine
but contains Uracil (‘U’) rather than
(‘C’).
Thymine.

Base Pairs Adenine and Thymine pair (A-T) Adenine and Uracil pair (A-U)
 Cytosine and Guanine pair (C-G) Cytosine and Guanine pair (C-G)

RNA forms in the nucleolus, and then

DNA is found in the nucleus, with a small moves to specialized regions of the
Location amount of DNA also present in cytoplasm depending on the type of
mitochondria. RNA formed.

Due to its deoxyribose sugar, which contains RNA, containing a ribose sugar, is
one less oxygen-containing hydroxyl group, more reactive than DNA and is not
Reactivity DNA is a more stable molecule than RNA, stable in alkaline conditions. RNA’s
which is useful for a molecule which has the larger helical grooves mean it is more
task of keeping genetic information safe. easily subject to attack by enzymes.

Ultraviolet
DNA is vulnerable to damage by ultraviolet RNA is more resistant to damage from
(UV)
light. UV light than DNA.
Sensitivity

Mutation

A mutation is a heritable change in the DNA sequence of an organism. The resulting organism,
called a mutant, may have a recognizable change in phenotype compared to the wild type, which
is the phenotype most commonly observed in nature. A change in the DNA sequence is conferred
to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on
translation.
RECOMBINANT DNA TECHNOLOGY

Recombinant rDNA technology involves procedures for analyzing or combining DNA fragments
from one or several organisms including the introduction of the rDNA molecule into a cell for its
replication, or integration into the genome of the target cell. The new cell have more value to
science, medicine, agriculture, and industry. The fundamental goal of laboratory geneticists is to
isolate, characterize, and manipulate genes.

Recombinant DNA is made from combining DNA from different sources. Image by Walter Suza.

Transformation of cells with rDNA produces organisms called bioengineered or genetically

modified organisms (GMOs). The GMOs contain new traits from another organism. The first
GMOs were Escherichia coli cells that were transformed with genes from human to produce
various proteins for pharmaceutical purposes.

Clone: is a group of individual cells or organisms descended from one progenitor. This means
that the members of a clone are genetically identical, because cell replication produces identical
daughter cells each time. cloning provided scientists with the ability to produce many copies of a
single fragment of DNA, such as a gene, creating identical copies that constitute a DNA clone. In
practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule
and then allowing this molecule to replicate inside a simple living cell such as a bacterium.
The small replicating molecule is called a DNA vector (carrier). The most commonly used
vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast
cells.

Steps in Recombinant DNA (rDNA) Technology

Recombinant DNA is composed of sequences that are derived from different sources. The
process to achieve this involves the following steps:

 Isolation of genetic material

 Cutting of DNA at specific locations
 Joining of DNA fragments by ligation and homopolymer tailing
 Insertion of DNA into the host cell
 Selection and screening of transformed cells

The last step can be achieved by an immunological method or nucleic acid hybridization, blue-
white screening or insertional inactivation.

Benefits Of Recombinant DNA Technology

Several benefits recombinant DNA technology aimed to improve the standard of living have
found its use in the following area including;
1. Genetically engineered proteins in therapeutics
2. Gene Therapy
3. In improved disease resistance variance in crops and improve yields in
agriculture.
4. Forensics science and crime detection
5. Improving our environment and keeping it pollution free
6. Improved generation of Transgenic Animals
Horizontal Gene Transfer (HGT).
There are ways horizontal gene transfer occur which include;

Conjugation

This 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.

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. Typicaly the process requires a donor cell that at some point lysed and 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.
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 transductions: generalized transduction and specialized transduction.