BACTERIAL TAXONOMY * Staphylococcus aureus: gram-positive coccus; Bacteremia or skin infection; Staphylo-bunch

or grapes, coccus-berry; Scottish surgeon Alexander Oregon 1880s- described in pus from
Microbial TAXONOMY knee joint abscess; German physician Friedrich Julius Rosenbach- differentiated color of
→ Science of orderly classifying living beings or organisms bacteria colonies, aureus as gold/yellow
(Taxon/taxa- arrangement ; nomos- Law)
→ Carl von Linne (also known as Linnaeus) – a Swedish 2. Nomenclature (naming)
botanist – rules for classification for classification and → assignment of scientific names to various taxonomic categories by certain dominant
established taxonomic categories (taxa s: taxon) (originated 250years ago) features.
→ area of biologic science comprising 3 distinct, but highly interrelated disciplines:
3. Identification
1. Classification → process of discovering and recovering the traits of organisms
→ orderly arrangement of organisms into a hierarchy or group(taxa), based on → microorganisms’ key features are delineated
similarities in morphologic, physiologic & genetic traits. → 2 methods how to identify bacteria:
→ Taxa designations: a) Phenotypic – observable physical & functional features
i. Kingdom (similar divisions/phylum) b) Genotypic – organisms’ genetic makeup
ii. Phylum (similar class)
iii. Class (similar order)  Phenotypic Criteria: MACROSCOPIC MORPHOLOGY
iv. Order (similar family) → microbial growth pattern on culture media
v. Family (similar genus) → isolated using solid agar; solidifying agents
vi. Genus (similar species)  Blood agar
vii. Species (basic taxa; collection of bacterial strains w/ common - used to grow fastidious organisms & differential media based on
physiologic & genetic features) hemolytic patterns. (5% shift blood or defibrinated blood)
* Subspecies = taxonomic group of species, divided based  Chocolate agar
on phenotypic differences. Designations include: - containing RBC that’s slowly heated to 80°C used for growing
a) Serotype fastidious respiratory bacteria RBC is lysed to a molten agar base
b) Biotype (same species, same genetic makeup but different physiologic  MacConkey agar plate
traits) - contains lactose, when fermented, turns pink
c) Genotype  Triple Sugar Iron Test
- contains 3 types of carbohydrates; sucrose, lactose, glucose
Example: Bacterial Classification

* Escherichia coli: gram-negative bacillus, facultative anaerobic group rods; from German-  Phenotypic Criteria: MICROSCOPIC MORPHOLOGY
Australian pediatrician Theodor Escherich; infant fecal sample; anaerobic culture method & → Size, shape, intracellular inclusions, cellular appendages & arrangements
gram staining → Average diameter of spherical bacteria:
 - .5µm-2µm ; For rod/filamentous bacteria: 1µm-10µm & .25µm-1µm  Phenotypic Criteria: ANTIGENIC AND SUBCELLULAR PROPERTIES
diameter → Profiling microorganisms by various serologic & immunologic methods
→ Bacterial Morphology: 3 bacterial shapes and arrangements → Establishment of molecular constituents (cell wall, cell membrane, enzymatic
 Coccus (spherical) contents & subcellular properties)
 Bacillus (rod) * H. influenzae type B: most serious Haemophilus, which leads to deadly brain infection,
 Spiral (twisted/curved to corkscrew) commonly associated w/ meningitis among young children.
 Latex Agglutination Principle:
→ Different Stains: - Coagglutination = uses antibody to increase visibility of
 Most common – Gram stain & Acid-fast stain agglutination reaction between antigen & antibody
 Special stains – Indian ink capsule stain & Flagellar stain (silver stain)  New Field Quellung Test = capsule swelling phenomena w/ type-specific
 Simple stains – Crystal violet stain & Methylene blue stain antisera
(Positive – capsule swollen; Negative- capsule not swollen)
 Phenotypic Criteria: ENVIRONMENTAL REQUIREMENTS (Pneumococcal Quellung Reaction = 1902 by Fred Newfield, applied only to
→ Ability of organisms to grow at various temperatures, presence of oxygen and other S. pneumoniae; a capsulated organism which is the most causative &
gases at various pH levels community acquired pneumonia)
→ Temperature:
 Psychrophiles (cool/cold); 4°C -25°C  Genotypic Criteria: DNA BASE COMPOSITION RATIO & NUCLEIC ACID BASE SEQUENCE
 Mesophiles (moderate); 20°C -45°C ANALYSIS
 Thermophiles (heat/hot); 50°C -80°C → Nucleic Acid Base Sequence Analysis
 Hyperthermophiles; 80°C -110°C - homologous, base sequence along w/ DNA & RNA strand
 Extreme Hyperthermophiles; 120°C - To purify DNA preparations, bacterial colonies and specimen (sputa tissue
→ pH: biopsies & pus secretion)
 Acidophile; pH of 1 or 2 - 5 E.g. Plasmid analysis, Southern blot analysis, Ribotyping, Restriction endonucleases
 Neutrophile; pH of 6.5-7 analysis, PCR amplification, etc.
 Alkaliphile; pH of 8 or 8.5 – 10 or 10.5
(pH of bacteria; 6.5-7.5 while pH of culture media; 7-7.5) CELL STRUCTURE

 Phenotypic Criteria: RESISTANCE PROFILE EUKARYOTIC CELLS

→ Resistance pattern for all isolates
 Nucleus (contains cell genome ‘genotype’)
→ Phenotypic Resistance
Nucleoplasm; gelatinous matrix
- Resistance w/out any genetic alteration Nuclear membrane; nuclear pores (selective permeability)
- None inherited resistance Chromosomes; linear DNA (double helix)
- Stationary growth phase or even persistents Nucleolus; site of ribosomal RNA synthesis
 Cytoplasmic Structures - Contains enzymes from Krebs Cycle (TCA) and fatty acid
Endoplasmic Reticulum (ER) cycles (DNA, RNA, ribosomes & calcium granules).
 network of membrane-bound channels - Final electron acceptor is oxygen
 highly convoluted system of membranes - Electron transport chain produces ATP called oxidative
 interconnected & arranged to form transport, network of tubules and phosphorylation
flattened sacs in the cytoplasm  Inner membrane
 2 types: - (enzyme -ATP synthase) transport proteins that regulate
 Rough ER = Eukaryotic 80S ribosomes (Prokaryotic 70S ribosomes) metabolites in & out of the matrix
Major producer of glycoproteins - Arranged to Cristae; an inner fold membrane, which
 Smooth ER = No ribosomes increases surface area for energy production via oxidative
Lipid synthesis & some carbohydrates phosphorylation (ADP is phosphorylated to ATP)
Golgi Complex/Apparatus/Body - Simple fission; has DNA & 70S ribosomes
 Only organelle named after discoverer; Camillo Golgi 1898
 Communicates w/ ER Plastids: Chloroplasts
 Stacked membranes to chemically modify & sort products of ER  Photosynthesis
 Proteins received are processed & transported to lysosomes, plasma  Only in plant cells
membranes or secretions  Contains green photo pigment called chlorophyll
 “packaging plants” helps process and package proteins & lipid molecules, to
be exported from cells Lysosomes & Peroxisomes
 Lysosomes
Mitochondria - contains degradative enzymes called lysozymes; w/ch breaks down
 “Powerhouse of the cell” foreign materials via phagocytosis & may destroy the entire cells
 Major site of ATP synthesis process called autolysis
 .75µm-3µm  Peroxisomes
 5 Roles: - contains oxidative enzymes called catalase; w/ch generates ATP
 Production of ATP production (prominent in mammalian liver cells)
 Store Calcium (calcium homeostasis)
 Regulation of innate immunity Cytoskeleton
 Apoptosis (store caspases)  3D structure that fills the cytoplasm or systems of fibers
 Stem cell regulation  3 primary fibers: (supports, strengthen & stiffen the cell)
 Structures:  Microfilaments (3nm-6nm diameter)
 Outer membrane - defining & maintaining cell shape; gliding/motility;
- (porins) Movement of ions, in& out of the mitochondrion contraction & cytokines
 Matrix  Intermediate filaments (10nm)
 - cells’ tensile strength  Cell envelope
 Microtubules (20nm-25nm)  Internal structures
- cell structure, motility & division, maintaining cell
structure via microfilaments to form spindle fibers for  Intracellular Structures: CYTOPLASM
separating chromosomes during mitosis → for cell growth, metabolism and replication.

 Plasma Membrane aka: Cell/Cytoplasmic/Cellular membrane  Intracellular Structures: NUCLEOID & BACTERIAL CHROMOSOMES
→ “skin” around the cell → bacterial chromosomal DNA (genophore lacks chromatin) is contained
→ Semipermeable lipid bilayer & protein transport (entering/exiting) → No membrane-bound nucleus
→ Organize & package chromosomes, similar to histones
 Cell Wall → Under light microscope via Feulgen Staining
→ Cell rigidity, strength & protection against mechanical stress → Visualized on electron micrograph at high magnification
→ Not found in animals & heterotrophic protists
→ Cellulose; cell walls of algae  Intracellular Structures: PLASMIDS
→ Chitin; cell walls of fungi → Small extra-chromosomal DNA
→ contains genes for antibiotic resistance or virulence
 Motility Organelles → provide a level of genetic flexibility
→ 2 basic structures: → from one bacterium to another via conjugation & transduction
 Cilia → Types of bacterial plasmid (Based on function)
- short, hair-like; only in eukaryotic cells; sweeping or pendular i. Fertility (F) plasmid
movement; locomotion, feeding & circulation  Conjugation
 Flagella - Transfer genetic information
- long, whip-like; both in eukaryotic & prokaryotic cells; undular - No cellular reproduction
movement; locomotion only - Pili/Fimbriae are responsible
(Both consists of microtubules, halo protein cylinders, tubulin, surrounded by a membrane. ii. Resistance (R) plasmid
The arrangement of microtubules is called 9 + 2 system; 9 peripheral pairs of microtubules, - R factor contains antibiotic or drug resistance gene
surrounding 2 singles central microtubules) iii. Virulence plasmid
- Host of plasmids or bacterium becomes pathogenic
PROKARYOTIC CELLS iv. Degradative plasmid
- Digestion of unusual substances (toluene or salicylic acid)
→ Most common: Bacteria & Archaea v. Col plasmid
→ Lack membrane-enclosed organelles - Genes that kill other bacteria (Bacteriocin – E. coli)
→ 3 categories:
 External structures  Intracellular Structures: RIBOSOMES (POLYSOMES)
 → Site of translation; Protein synthesis → Movement:
→ Polysomes are ribosomes bound to mRNA that synthesize proteins  Passive
→ Ribosomes are constructed from proteins w/ RNA - occur w/out cellular energy
→ Antibiotics interfering with translation causes faulty protein synthesis  Active
→ Sedimentation rate of 70S - requires the cell to expend energy
→ Translate genetic code to amino acid  Group translocation
→ Antibiotics: - moved w/o change in structure
 Streptomycin
- stops protein synthesis via 30S subunit (e.g. Tuberculosis)  Cell Envelope: CELL WALL
 Tetracycline → outer covering of most cells
- kills gram-positive & gram-negative organisms via 30S subunit → protects the bacterial cell from osmotic lysis and gives it shape
 Chloramphenicol → Major component of bacterial CW:
- binds to 30S subunits  Peptidoglycan (murein/mucopeptides) = [polysaccharides + proteins]
 Linezolid → composed of long chains of alternating molecules of N-acetylglucosamine (NAG) and
- for gram-positive organisms, binding to initial step process of N-acetylmuramic acid (NAM)
protein synthesis → anchor appendages like the pili & flagella and protrude through the wall to the
outside
 Intracellular Structures: INCLUSION BODIES → Target site of some antibiotics:
→ Inclusions: non-living components of the cell (e.g. Penicillin = kills bacteria via binding beta lactam ring to DD-transpeptidase,
→ Vesicles: rigid gas-filled vacuoles (common in aquatic photosynthetic bacteria) inhibiting cross-linking activity and prevent new cell formation)
→ Serve as important identification characters for bacterial pathogens → Gram Staining (Most common differential stain) 2 Major Classes:
* Ex: Corynebacterium diphtheriae (metachromatic granules or volutin) = Babes Ernst bodies,  Gram Positive (dark blue or violet)
used to degrade and as source of phosphates for nucleic acid & phospholipids synthesis - Thicker
- Contains peptidoglycan
 Cell Envelope: PLASMA (CELL) MEMBRANE - Lipid content is low, decolorizing the cell and its cell wall dehydrates
→ regulates the flow of substances in and out of the cell (selective permeable & shrinks, w/ch closes the pores to prevent the stain from exiting
membrane)
→ Site of many metabolic reactions such as respiration, fermentation, and  Gram Negative (pink)
photosynthesis - Thinner peptidoglycan layer
→ excretion of hydrolytic enzymes and pathogenicity proteins and chemotactic agents - Has lipid component that dissolves w/ CV Iodine complex wash due
→ Phospholipid bilayer to decolorization process
- surrounding the cytoplasm - Added w/ alcohol or acetone: CVI complex leach out & unstained
- Formed w/ protein linkage
- Ability to move in membrane place
 - Added w/ counterstain: CVI complex is accepted (gram-positive will → NO cell walls
not accept or receive) → Lacks target for cell-wall inhibiting agents (resistant to penicillin)

 Cell Envelope: OUTER MEMBRANE  External Structures: FLAGELLA

→ Second lipid bilayer → act like propellers; to help bacteria move towards nutrients, away from toxic
→ Less permeable than plasma membrane chemical or towards the light
→ Composed of lipopolysaccharides [LPS]; acts as endotoxin → Undular movement
→ 3 structures: → Made up of protein subunits called flagellin
 O antigen (O polysaccharide) → Basal region/body is embedder in the plasma membrane
- Outermost structure → Arrangements:
- Triggers immune response  Monotrichous – single flagellum
 Core polysaccharide  Amphitrichous – flagellum at each end
- Both Core polysaccharide & Lipid A anchors the polysaccharide into  Lopotrichous – clusters of flagella at the poles of the cell
the outer membrane  Peritrichous – flagella distributed over the entire surface of the cell.
 Lipid A (endotoxin) → Hook region connects the basal body to the filament
- Large amount can trigger toxic shock, causing body wide (Gram-Positive: 2 basal body rings
inflammatory response Gram-Negative: 4 basal body rings)
→ Lipopolysaccharides:
- stabilize outer membrane  External Structures: FIMBRIAE & PILI
- block access to other parts of the cell wall → Fimbriae
- host response to pathogenic gram-negative bacteria - short bristle-like proteins
(e.g. E. coli 0157 – O side chain detect a specific strain of E. coli via serologic or - attach to surfaces and to other cells
immunologic tests; primary cause of hemolytic uremic syndrome, w/ch is a deadly → Pili
bacterium that causes diarrhea & kidney failure) - longer, less numerous protein
- aid in attachment to surfaces
 Cell Envelope: ACID FAST CELL WALL - w/out pile, bacteria can’t infect or attach to the host or solid surfaces
→ Contain large amounts of waxes (mycolic acids linked to arabinogalactan) * F pilus or sex pilus - transfer of DNA between bacterial cell [Conjugation]
→ Noted in tubercle bacillus (found in mycobacterium spp.) * Type IV pili - generate movement)
→ Hydrophobic structure that renders bacteria resistance
[Why acid-fast? If a dye is introduced into these cells by brief heating or treatment  External Structures: GLYCOCALYX
with detergents, it cannot be removed by dilute hydrochloric acid, as in other → Sticky coating
bacteria] → protection, attachment to surfaces and formation of biofilms
→ 2 forms:
 Cell Envelope: MYCOPLASMAS  Capsule
 - highly organized rigid structure - the cell must grow over its entire surface until the time of cell
- polysaccharide layer that envelopes the cell division, when a new hemispherical pole forms at the division
- enhance ability of bacterial pathogens to cause disease septum in the middle of the cell
- provide protection from phagocytosis  For the cell to divide in half;
- attachment to the surfaces - the peptidoglycan structure must be different in the hemispherical
 Slime layer cap than in the straight portion of the cell wall, and different wall-
- Same as capsule; But is a disorganized loose structure crosslinking enzymes must be active at the septum than elsewhere

BACTERIAL METABOLISM, GROWTH, & GENETICS

 External Structures: ENDOSPORES
→ Dormant, tough, non-reproductive structure Bacterial Metabolism - involves all the steps in consuming something and using the energy from it
→ Germination -> vegetative cells to make the building blocks needed to survive and replicate. A metabolite is any molecule that is a
→ Resistance: nutrient, an intermediary product, or an end product in a metabolic reaction. Most metabolic
reactions fall into two categories: catabolism and anabolism. This section focuses only on the
 UV and Gamma radiation
metabolism typical of medically relevant bacteria.
 Desiccation
 Lysozyme Four processes of Bacterial Metabolism
 Temperature
1. Fueling is considered the utilization of metabolic pathways that are involved in the acquisition of
 Starvation
nutrients from the environment, production of precursor metabolites, and energy production.
 Chemical
→ Bacillus and Clostridium (gram-positive) l . l . Acquisition of nutrients - nutrients must cross the bacterial cell wall and membrane
→ Killed using autoclaving a. Diffusion - Water, Oxygen, and Carbon dioxide
→ disinfectants b. Active transport - certain sugars, most amino acids, organic acids, and many inorganic ions
→ Relationship: enter the bacterial cell this way. This action is mediated by an energy-dependent pump and
 Sporulation involves carrier molecules found in the bacterial cell membrane. These carrier molecules
- Endospore formation transport the nutrients into the cell.
c. Group translocation - It is also energy-dependent but the nutrients undergo chemical
- Deflation of some nutrients (carbon, nitrogen, phosphorus) and
modification before they can enter the cell. Many sugars, purines, pyrimidines, and fatty acids
form spores
are transported by this mechanism.
 Germination
1.2. Production of precursor metabolites - As the nutrients enter these are processed to become
- Return to vegetative state precursor metabolites. The precursor metabolites are listed below:

 Cellular Division: BINARY FISSION a. Glucose 6- g

→ Most bacteria divide by binary fission (asexual reproduction) into 2 equal progeny phosphate Phosphoenolpyruvate
b. Fructose 6- h. Pyruvate
cells:
phosphate
 For this process to occur;
c. Pentose 5- i. Acetyl COA
phosphate
 d. Erythrose 4- j. Succinyl COA
phosphate
a. 3-Phosphoglycerate k. Oxaloacetate
b. F-Ketoglutarate
These precursor metabolites are processed through two central pathways, and two alternative
pathways:
PATHWAYS CHARACTERISTICS
Embden- Major metabolic pathway; It is an anaerobic
Meyerhof- process; In glycolysis, a sixcarbon molecule of
Parnas Pathway, glucose is ultimately broken down into two three-
Embden- carbon molecules of pyruvic acid (also called
Meyerhof or pyruvate).; provides only a little amount of energy
Glycolytic producing a net yield of two ATPs; It happens in the
pathway c to lasm of the cell
Tricarboxylic Acid
Major metabolic pathway; The pyruvic acid
Cycle (TCA), Citric
molecules produced during glycolysis are converted
Acid or into acetyl-coenzyme A (acetyl-CoA) molecules,
Krebs cycle which then enter the Krebs cycle; . In the first step
of the Krebs cycle, acetyl-CoA combines with
oxaloacetate to produce citric acid (a tricarboxylic
acid); It is referred to as a cycle because at the end
of the eight reactions, the biochemical pathway
ends up back at its starting point— oxaloacetate.
Only two ATP molecules are produced during the
Krebs cycle, but several products (e.g., NADH,
FADH2, and hydrogen ions) that are formed durin
the Krebs c cle enter the electron trans ort chain.
Entner-Doudoroff Alternative pathway; catalyzes the degradation of
gluconate and glucose. The gluconate is
phosphorylated, dehydrated, and converted into
pyruvate and I ceraldeh de, leadin to ethanol
roduction
Pentose Alternative pathway; uses glucose to produce
reduced nicotinamide adenine dinucleotide
phosphate phosphate (NADPH), pentoses, and tetroses for bios
pathway thetic reactions such as nucleoside and amino acid s
thesis.
I .3 Energy Production
a. Substrate-level phosphorylation, high-energy phosphate bonds produced by the central
pathways are donated to adenosine diphosphate (ADP) to form ATP directly from the
substrate as opposed to generation via the electron transport chain. Pyruvate, a primary
intermediate in the central pathways, serves as the initial substrate for several other pathways
to generate ATP by substrate-level phosphorylation. Other pathways constitute the
fermentative metabolism which does not require Oxygen and produces various products such
as alcohols, acids, Carbon dioxides, and Hydrogen.
b.Electron transfer/ Oxidative phosphorylation, involves an electron transport system that
conducts a series of electron transfers from reduced carrier molecules such as NADH2 ,
NADPH2 and FADH2 flavin adenine dinucleotide), produced in the central pathways, to a
terminal electron acceptor. The energy produced by the series of oxidation-reduction
reactions is used to generate ATP from ADP.
b. 1. Aerobic respiration - when Oxygen is used as a terminal electron acceptor
b.2. Anaerobic respiration - uses final electron acceptor other than Oxygen
1. Biosynthesis
In anabolic metabolism, precursor compounds are joined for the creation of larger molecules
(polymers) required for assembly of cellular structures. Biosynthetic processes use the
precursor products in dozens of pathways to produce a variety of building blocks, such as
amino acids, fatty acids, sugars, and nucleotides.
2. and 4. Polymerization and Assembly
Anabolic reactions assemble (polymerize) the building blocks into macromolecules, including
lipids, lipopolysaccharides, polysaccharides, proteins, and nucleic acids. This synthesis of
macromolecules is driven by energy and enzymatic activity in the cell. Cellular structures are
the product of all the genetic and metabolic processes.
Bacterial Growth refers to the bacterial multiplication and proliferation. Bacteria divides by binary
fission producing two daughter cells. On a culture medium, binary fission continues through many
generations to form a colony. A bacterial colony is a mound or pile of bacteria containing millions of
cells. Generation time (doubling time) refers to the time it take for one bacterial cell to divide via
binary fission into two daughter cells. In the laboratory, Escherichia coli, Vibrio cholerae, and
Staphylococcus spp. all have a generation time of about 20 minutes. Pseudomonas and some
Clostridium spp. May divide every 10 minutes. Mycobacterium tuberculosis may divide only for 18-
24 hours. Bacteria with short generation time are called rapid growers while those with long
generation time are slow growers. The growth of microorganisms are dependent on temperature,
pH, moisture content, available nutrients, and the characteristics of other organisms present. Some
bacteria require complex nutritional requirements and are called "fastidious." Some organisms will
not grow on artificial culture media because they are obligate intracellular like rickettsias,
chlamydias, Treponema pallidum, and Mycobacterium leprae. Obligate intracellular microorganisms
only grow when inoculated into embryonated chicken egg, cell cultures, or live animals.
Three major nutritional needs of all bacteria:
1. Carbon - 50% of the dry weight of a bacterium is Carbon; used for making cellular
constituents
2. Nitrogen - for making proteins and nucleic acids ; makes up of 14% of a bacterium's
dry weight
3. A source of ATP - for performing cellular functions.
4% of the bacterium's dry weight is made up of phosphate for nucleic acid synthesis,
phospholipids of cell membranes, and sulfur for protein synthesis v/ Na+, K+. Cl-, and Ca++ are also
required
Two basic groups of mirobes based on how they meet their nutritional needs:
1. Autotrophs (Lithotrophs) - Carbon dioxide is their main source of Carbon
1.1 Phototrophs -these are autotrophs that obtain energy photosynthetically
1.2 Chemolithotrophs - these are autotrophs that obtain energy by oxidation of inorganic
compounds
2. Heterotrophs - require more complex substances for growth; requires glucose as organic
source of Carbon and obtain energy by fermenting or or oxidizing organic substances; all
bacteria that inhabit the human body are heterotrophs
Phases of bacterial growth (see Figure 1)
1. Lagphase v/ period where the organism is adjusting into its new environment v/ little or
no cell division happens
Can last for 1 hour or several days
The organism is undergoing a a period of intense metabolic activity, in particular synthesis of
enzymes
2. Log or Exponential Phase v/ Bacterial cells divide and enter a period of growth or
logarithmic increase v/ Cellular reproduction is most active.
Cell is most metabolically active. Generation time is
constant.
v/ Phase where the organism is most susceptible to antibiotics.
3. Stationary phase/Plateau/Equilibrium
Growth ceases because nutrients are depleted and waste products are
accumulated The number of dead cells are equal to that of the viable cells.
4. Death or Logartithmic Decline Phase
The number of deaths is greater than the number of new cells.
Total bacterial count is still the same, only viable (living) cells are decreased.
After majority of the cells have died, death rate decreases causing some cells to survive
Viable But Not Culturable Cells (VBNC) occur as genetic response of the bacteria triggered by
starving in the stationary phase (Some bacteria may form spores in order to survive while
some become dormant for years.)
VBNC resumes growth when appropriate conditions are available.
THE GIST OF MICROBIAL GENETICS
This topic will only be limited to bacterial genetics. Archaea will also be mentioned since these two
are considered to be Prokaryote siblings, although archaea is much more similar to eukaryotes. The
objectives of this topic are the following: (a) Seeing how genetic material is organized, (b) Finding
out about transcription and translation, (c) Fine-tuning the amount and activity of proteins, and (d)
Keeping track of changes to the genome.
The Central Dogma of Life
In all cells, the instructions within the DNA are first copied into RNA, which is then used to make
protein. The central dogma of biology is then a flow of information from DNA to protein, through
RNA, like this:
DNA -9 RNA -9 Protein
The basic unit of heredity if called genes. Genes contain instructions for protein synthesis that exist
as strings of DNA. All the genes of an organism or the genetic makeup is collectively called genome.
Phenotype refers to the organism's structural and physiologic properties which is the target of
traditional genetics. While phenotype is the organism's observable characteristics, genotype on the
other hand refers to the set of genes that the organism carries. Resistance of an organism is an
example of a phenotype while the genes and their chemical interactions that make the organism
resistant to antibiotics refers to genotype.
DNA: The Formula of Life
Deoxyribonucleic acid (DNA) is the fundamental nucleic acid of heredity. Its building blocks is called
a nucleotide which is composed of: (a) a Nitrogenous base, (b) a pentose sugar, and; (c) a
phosphate group which are covalently bonded together.
The Nitrogenous bases are of two types: The first type is the six-membered heterocyclic ring called
Pyrimidine. Thymine and Cytosine belong to this group of Nitrogen base (Uracil, a pyrimidine found
in RNA replaces Thymine in the RNA structure). The second type has a fused heterocyclic ring called
Purine. Adenine and Guanine are purines. The pentose sugar deoxyribose is bonded together
nitrogenous bases in a covalent N-glycosidic bond. Specifically, the 1 st Carbon of deoxyribose is
bound to the 1 st Nitrogen of a pyrimidine, and another deoxyribose is bound to the 9 th Nitrogen of a
purine. (See Figure 2). This structure is called the Nucleoside.
DNA molecules are double-stranded with complementary bases. Such that according to the
discovery of
Erwin Chargaff, a purine must pair with a pyrimidine. In DNA, the complementary bases are Adenine
and Thymine, and Guanine and Cytosine. (See Figure 3). The complementary bases are bonded
together by weak Hydrogen bonds. Two hydrogen bonds for A-T pairing, while three for the G-C
pair.
The complementary strands exhibit anti-parallelism. Such that one strand runs in 3' to 5' direction
while the other runs in a 5' to 3' direction. (See Figure 4). The complementarity of the bases allows
one strand to be the template strand which provides the information for replication to make a
coding strand (new strand formed from copying the template strand). The base pairs are stacked
within the center of the DNA double helix (see Figure 5) and they determine its genetic information.
The DNA molecule is expressed in thousand base pairs of kilo-base pairs (kbp).
The Structure of RNA
RNA most frequently occurs in single-stranded form. The base uracil (U) replaces thymine (T) in
DNA, so the complementary bases that determine the structure of RNA are A-U and C-G. Ribose is
the pentose sugar present in this type of nucleic acid. There are three classes of RNA namely
messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). The most general
function of RNA is communication of DNA gene sequences in the form of messenger RNA (mRNA) to
ribosomes. These processes are referred to as transcription and translation. mRNA (referred to as
+ssRNA) is transcribed as the RNA complement to the coding DNA strand. This mRNA is then
translated by ribosomes. The ribosomes, which contain both ribosomal RNA (rRNA) and proteins,
translate this message into the primary structure of proteins via aminoacyl-transfer RNAs (tRNAs).
(See Figure 6). Bacterial chromosomes have 3 types of rRNAS. These are 23S (2.9 kbp), (0.12 kbp),
and 16S (1.54 kbp).
The Bacterial Genome
The genomes of bacteria and archaea are, for the most part, arranged as a single (haploid) circular
chromosome and some extra-chromosomal genetic material, called plasmids. A few bacteria (eg,
Brucella melitensis, Burkholderia pseudomallei, and Vibrio cholerae) have genomes consisting of
two circular DNA molecules. Many bacteria contain additional genes on plasmids that range in size
from several to 100 kbp.
Replicons which are composed of bacterial chromosomes and plasmids contain genetic information
necessary for their own replication. The chromosome contains all the essential genes required for
life, whereas plasmids contain useful but not strictly essential genes. There are 2 important groups
of genes in bacteria. Some bacteria possess the "Pathogenecity islands." These genes code for
pathogenic determinants that make a bacterium efficient in causing a disease. Genes that code for
antibiotic resistance, adhesins, invasins, and exotoxins are examples of pathogenic determinants.
The second group of genes is called the "Housekeeping genes" which are essential for bacterial
growth. These genes are carried by the bacterial chromosome and some specialized functions that
are not encoded in the bacterial chromosome are contained in plasmids. For example, the
degradation of camphor, toluene, octane, and salicylic acid by Pseudomonas aeruginosa is a
metabolic activity determined by plasmids.
To remember which Nitrogen bases are purines and pyrimidine, here are helpful
mnemonics for you.
"PURe As Gold." (PURine Adenine Guanine)
"CUT the Pie." (Cytosine, Uracil, and Thymine PIErimidine (Pyrimidines))
''Y do U have one ring?" (There is Y in the word pyrimidine and so are the two pyrimidine
bases namely, Cytosine and Thymine. The U is for the pyrimidine Uracil which is only
found in RNA. The 3 pyrimidine bases have one ring in their structures.)
 To remember the base pairings and the number ofHydrogen bonds per pair, these d. Topoisomerases - assist DnaB in unwinding the helix
mnemonics may help you. e. Single-stranded binding proteins (SSB) - keep apart the single strands and protect ssDNA
segments from attack by nucleases.
"Meet me AT 2 pm." (Adenine-Thymine, 2 Hydrogen bonds)
2. Elongation
"There are onl 3 eo le in the GC." 3 H dro en bonds for Guanine-C osine Replication forks have leading and lagging strands:
Bacterial Replication
Leading strand - proceeds continuously in the 5' to 3' direction; requires a single RNA primer for
The replication of bacterial DNA (bacterial chromosome and plasmids) is usually bidirectional (see the DNA polymerase to initiate DNA synthesis.
Figure 7). It begins at one point and moves in two directions. Replication is semiconservative such
that the two old strands become templates to synthesize new daughter strands. A new strand is Lagging strand - requires discontinuous 5' to 3' synthesis through the formation of short Okazaki
formed at the replication fork (where two strands are separated). Some bacterial plasmids may fragments; requires multiple RNA primers for the DNA polymerase to initiate synthesis of the
have as many as 30 copies in one bacterial cell, and mutations causing relaxed control of plasmid Okazaki fragments.
replication can result in 10-fold higher copy numbers. The replication of circular double-stranded Lagging strand synthesis: The primosome travels along the lagging strand and stops and reverses
bacterial DNA begins at the ori locus and ends at the ter (termination) region. direction at intervals to synthesize a short RNA primer. Subsequently, pol Ill synthesizes DNA
Other plasmids also replicate unidirectionally or "rolling circle replication." One important feature beginning at the 3' end of the primer. As lagging strand synthesis continues, the RNA primers are
of plasmids is that they can be transferred between bacteria. For the most part, this happens in two removed and replaced by DNA segments synthesized by pol I. DNA ligase joins the Okazaki
ways: (a) Bacteria die releasing their plasmids and other bacteria take them up, and (b) Plasmids, or fragments.
other genetic material, are actively transferred from one bacterium to another by a process called 3. Termination v/ Replication ends when the replication forks meet on the other side of the
conjugation. For the former to happen, the recipient bacterium must be naturally competent (able circular chromosome at the termination site, the ter region.
to take up DNA naturally from its environment). For the latter, some plasmids are conjugative v/ Then the 2 daughter DNA molecules separate.
plasmids which are capable of transferring themselves, other bacterial plasmids, and even
DIFFERENCES IN DNA REPLICATION IN PROKARYOTES AND EUKARYOTES
chromosomal DNA between bacteria. Other plasmids include, resistance plasmids which carry
Proka otes Eukar otes
genes for resistance to antibiotics, heavy metals, and other cellular defenses; Virulence plasmids
1. Five polymerase (1, 11, 111, IV, V) Five polymerases (u, ß, ö, c)
that carry virulence factors, and; Colicin plasmids which carry bacteriocins used to inhibit or kill Functions Functions a-polymerizer ß-repair
I — synthesis, proofreading, repair & removal of RNA y - replicates mitochondrial DNA
other bacteria. Bacteriocins have a narrower range than antibiotics, so they're specifically targeted primers Il — repair ö-main polymerizer c-unknown
to particular bacteria. Ill — main polymerizer
IV & V — repair under unique conditions

Steps in Bacterial Replication (see Figure 8): 3. polymerases are also exonucleases Not all polymerases are exonucleases
4. one ori in & re lication Several ori ins of re lication
1. Initiation v/ formation of a "bubble" which corresponds to the local unwinding of the DNA 5. okazaki fragments (1000-2000 residues long) 150-200 residues long
helix at the origin of DNA replication (oriC). The point at which the parental DNA is being unwound, 6. no proteins complexed to DNA Histones complexed to DNA
and the new DNA strands are being synthesized, is called the replication fork. Enzymes and Proteins Gene Expression (Transcription and Translation) see Figure 9
involved:
a. DnaA protein - binds to four 9-bp sites within the oriC sequence. The formation of the A. Transcription

DnaADNA complex causes the "melting" of the DNA duplex in three nearby 13-bp sequences Gene expression begins with transcription. During transcription the DNA base sequence of the
(A-T rich). gene (i.e., the genetic code) is converted into an mRNA molecule that is complementary to the
b. DnaB protein - a helicase that unwinds the helix. gene's DNA sequence. Usually only one of the two DNA strands (sense strand) encodes for a
c. DnaC protein - forms a complex with DnaB functional gene product. This same strand is the template for mRNA synthesis. RNA polymerase
 is the enzyme central to the transcription process. The enzyme is composed of four protein
subunits and a sigma (s) factor. Sigma factors are required for the RNA polymerase to identify
the appropriate site on the DNA template where transcription of mRNA is initiated. This
initiation site is also known as the promoter sequence. The remainder of the enzyme functions
to unwind the dsDNA at the promoter sequence and use the DNA strand as a template to
sequentially add ribonucleotides (ATP, GTP, UTP, and CTP) to form the growing mRNA strand.
Transcription proceeds in a 5' to 3' direction. However, in mRNA, the Thymine of DNA is replaced
with Uracil. Termination of transcription may be facilitated by a rho (a prokaryotic protein)
cofactor or an intrinsic termination sequence. In bacteria, the mRNA molecules that result from
the transcription process are polycistronic; that is, they encode for several gene products.
Polycistronic mRNA may encode several genes whose products (proteins) are involved in a single
or closely related cellular function. When a cluster of genes is under the control of a single
promoter sequence, the gene group is referred to as an operon. The transcription process not
only produces mRNA but also tRNA, rRNA, and regulatory ncRNA molecules.
B. Translation
The next phase in gene expression, translation, involves protein synthesis. Through this
process the genetic code in mRNA molecules is translated into specific amino acid sequences
that are responsible for protein structure and function. The Genetic Code is used to translate
the genetic message from mRNA to protein. Features of the Code include the following:
A. Triplet
- a sequence of 3 bases (codon) specifies one amino acid
B. Non-overlapping - no bases are shared between consecutive codons
C. Commaless - no intervening sequences exist between codons
D. Degenerate - more than one triplet can code for the same amino acid
E. Universal - the same code is used by all organisms (prokaryotes, eukaryotes, viruses) with
few exceptions
The steps in protein synthesis are the following:
l . Initiation begins with the association of ribosomal subunits, mRNA, formylmethionine (f-
met) tRNA (carrying the initial amino acid of the protein to be synthesized), and various
initiation factors. Assembly of the complex begins at a specific 3- to 9-base (Shine-Dalgarno
sequence) on the mRNA about 10 bp upstream of the AUG start codon. After the initial
complex has been formed, addition of individual amino acids begins.
1.Elongation involves tRNAs and a host of elongation factors that mediate the sequential
addition of amino acids in a specific sequence dictated by the codon on the mRNA molecule As
the mRNA molecule threads through the ribosome in a 5' to 3' direction, peptide bonds are
formed between adjacent amino acids. Because multiple proteins encoded on an mRNA strand
can be translated at the same time, multiple ribosomes may be simultaneously associated with
one mRNA molecule. Such an arrangement is referred to as a polysome; its appearance
resembles a string of pearls.
2.Termination, the final step in translation, occurs when the ribosomal A site encounters a
stop or nonsense codon that does not specify an amino acid (i.e., a "stop signal").After
termination, most proteins must undergo modification, such as folding or enzymatic trimming, so
that protein function, transportation, or incorporation into various cellular structures can be
accomplished. This process is referred to as posttranslational modification. Genetic Exchange and
Diversity: Transfer of DNA
A. Restriction Enzymes
Restriction enzymes (restriction endonucleases) provide bacteria with a mechanism to distinguish
between their own DNA and DNA from other biologic sources. These enzymes hydrolyze (cleave)
DNA at restriction sites determined by specific DNA sequences ranging from 4 to 13 bases. A direct
biologic consequence of restriction can be cleavage of donor DNA before it has an opportunity to
become established as part of a recombinant replicon, rendering the bacterial "immune" to
incoming DNA.
B. Mechanisms of Recombination
Donor DNA which does not carry a gene for its own replication must establish itself into a recipient
strain by recombining.
B. l. Homologous recombination - the transfer of DNA from closely similar donor and recipient
(recombining genes of the same ancestry). This process of recombination requires a set of
genes known as rec. Homologous recombination is reciprocal. Meaning to say, the
introduction of donor gene into the recipient is mirrored by transferring of homologous gene
from the recipient to the donor.
B.2. Nonhomologous recombination - is achieved via enzymes that catalyze
recombination between two dissimilar DNA sequences. The recombination depends on the
enzyme brought by the donor DNA. For example, the integration of a transposon to a
recipient DNA for it to be replicated by the recipient strain. Gene conversion or the non-
reciprocal transfer makes the bacteria genetically diverse microorganisms.
C. Mechanisms of Gene Transfer
C.I. Conjugation- When cells transfer genetic material with the help of a conjugative plasmid.
 C.2. Transduction - Happens through the action of a virus that infects one bacterial cell
and transfers genes to another bacterial cell. Viruses that infect bacteria are called
bacteriophage or simply phage.
C.2. l. Generalized transduction - happens when a phage packages any genes present in a
bacterial cell, whether they're chromosomal, plasmid, or viral.
C.2.2. Specialized Transduction - occurs when only some bacterial genes can be
packaged into a phage, such as specific toxin genes or other virulence factors.
C.3. Transformation - When competent cells take up DNA from their environment
C.4. Transposition - is when a small piece of DNA, called a transposon,moves from one
location to another within the DNA of an organism. For this to happen, there has to be an
enzyme capable of cutting and resealing DNA and recognition sites where the enzyme acts.
There are simple and complex transposons but all cause DNA modifications.
I. Mutations
A. Point mutations - errors that occur during DNA replication that alter the sequence by one or
a small number of bases by adding too many nucleotides, too few nucleotides, or a wrong
nucleotide.
A. I. Silent mutations - when base substitutions do not have an effect to the final protein
product
A.2. Missense mutations - wrong nucleotide is added within the coding region of a
protein gene and that changes the amino acid that is incorporated into the resulting protein.
or "transfer" their DNA and make other bacteria also resistant to antibiotics. Other mobile elements
that can cause bacteria-to-bacteria transfer of resistance genes include transposons which contain
genes including those that facilitate their transfer from one locus to another causing most of
insertion mutations among bacteria. Transposons do not carry gene for their own replication and
are therefore dependent of their physical integration with a bacterial replicon. Complex
transposons carry gene for antibiotic resistance and are flanked by insertion sequences. So when
these insertion sequences (IS) insert/integrate themselves to the bacterial chromosome and/or
plasmid antibiotic resistance may therefore be passed horizontally or vertically. Another mobile
element is the bacteriophage. Bacteriophage is a virus that infects bacteria. Phages can assemble
part of their host's (bacterium) genetic material, including antibiotic resistance genes, causing
dissemination of antibiotic resistance as they infect multiple bacteria.
Bacteria also can fight antibiotics in other ways:
1. Natural selection - those bacteria that are more susceptible to antibiotics will die quickly,
leaving any surviving bacteria to pass on their resistant features to succeeding generations.
2. Biological Mutations - Since bacteria are extremely numerous, random mutation of bacterial
DNA generates a wide variety of genetic changes. Through mutation and selection, bacteria can
develop defense mechanisms against antibiotics. For example, some bacteria have developed
biochemical that can remove an antibiotic before it reaches its target, while others have evolved to
produce enzymes to inactivate the antibiotic.
3. Rapid reproduction - Bacteria reproduce rapidly, sometimes in as little as 20 minutes.
Therefore, it does ot take long for the antibiotic-resistant bacteria to comprise a large proportion of
a bacterial population.

A.3. Nonsense mutation - is the result of the wrong nucleotide being added within the
coding region of a protein gene, creating a stop codon. The resulting protein will then be
shorter than it is supposed to be, or truncated.

A.4. Frameshift mutations - is caused by the loss or addition of a nucleotide that changes
how the pattern of three nucleotides per codon is read. The result is that, from this point
forward, completely different amino acids may be incorporated into the protein.
"Antibiotic Resistance"

Often, antibiotic resistance genes are encoded in plasmids. Because some bacteria with
resistance genes encoded in their plasmids can transfer these genes to other bacteria, they make
these bacteria resistant and they can pass these genes to their progenies (vertical transfer) as they
replicate or to other bacteria (horizontal transfer). This simply means that some bacteria can share
 LIST OF FIGURES

Figure 1. The Bacterial Growth Curve Figure 3. The DNA Base Pairing by Erwin Chargaff

DNA Nucleotide
Phosphate One strand of
DNA goes from
5' to 3' (sugars) The other
strand is opposite in direction
going
3' to 5' (sugars)
Figure 4. The Anti-parallel DNA strands

6roup
Figure 2. The DNA Nucleotide
 Figure 7. Bidirectional bacterial replication

Figure 5. The DNA Double Helix

Stop

Coding strand
DNA
Template strand Nucleotides

RNA polymerase

RNA transcript 5'

Step 3: DNA polymerase moves along the ONA template

Figure 8. Basic Steps in DNA Replication

C-terminal Protein
DNA coding
strand
Figure 6. Transcription and Translation processes
 Compiled by: Rolaine Ann A. Polo, RMT, MPH

CT
ATT CAG CAT
T
GAA AAA CAA TAA GTC G TA
...3'
...5'
RNA DNA
Transcription polymerase template

3' CU I- IJUU AUU

UU AG CAU
J G C Ribosomes,tRNA,
cofactors

mRNA

H2N his COOH

Polypeptide
Figure 9. Overview of Gene Expression (Transcription and Translation)

References

1. Brooks, G. F., Jawetz, E., Melnick, J. L., & Adelberg, E. A. (2013). Jawetz, Melnick & Adelberg's medical microbiology (26th edition.). New York :
London: McGraw-Hill Medical ; McGraw-Hill
2. Centers for Disease Control and Prevention (2020). How Antibiotic Resistance Happens. Retrieved from
https://www.cdc.gov/drugresistance/about/how-resistance-happens.html
3. Karki, G. DNA Replication (2021). Online Biology Notes. Retrieved from https://www.onlinebiologynotes.com/dnareplication/.
4. Lumenlearning: Mechanismsof Microbial Genetics. Retrieved from
https://courses.lumenlearning.com/microbiology/chapter/ dna-replication/
5. Mahon, C. R., & Lehman, D. C., (2019). Textbook of Diagnostic Microbiology (6th edition). Elsevier Inc.
6. Missouri Department of Health Services: What is Antibiotic Resistance. Retrieved from https://
health.mo.gov/safety/antibioticresistance/generalinfo.php
7. Stearns, J. C., Kaiser, J. & Surette, M. G. (2019). Microbiology for Dummies. John Wiley & Sons, Inc., Hoboken, New Jersey
8. Tille, Patricia M., (2017). Bailey & Scott's diagnostic microbiology (14 th edition). St. Louis, Missouri :Elsevier, 9. Tortora, G. J., Funke, B. R., & Case,
C. L. (2013). Microbiology: An introduction (12th edition). Pearson Inc., USA