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Microbiology Basic and Applied
Dr. Bipinraj N K
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Role of Microbiology
Medical Industrial Molecular Biology Environmental Genetics & Recombinant DNA Tech
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Microorganisms or Microbes
Microscopic organism
Bacteria
Fungi
AlgaeProtozoa
Virus
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General Characteristics of Bacteria A prokaryotic microorganism(no membrane-enclosed
nucleus) Size: average size 0.5 m No mitochondria or chloroplasts Single chromosome
A closed circle of double-stranded DNA (Plasmid) Flagella may present (made up of protein flagellin) Ribosome present Rigid cell wall made of peptidoglycan. (Gram + and -) The plasma membrane : phospholipid bilayers Reproduction : asexual by fission or spore formation Sexual by conjugation
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Bacterial Cell Wall :G +Teichoic acids, polymersglycerol or ribitol joined bphosphate group
It has negative charge
80 nm
60 nm
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Bacterial Cell Wall : G -
8 nm
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Fungi Eukaryotic organism Unicellular (yeast) and
multicellular (mushrooms) Chitin in the cellwall Heterotrophic & no
chloroplast Reproduction by sexual &
asexual
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Cell structure Single cellualr: yeast Multicellualr : mold
Long, branched, threadlikefilaments of cells called
hyphae Septate Ceonocytic
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Dimorphism Dimorphic fungi can change from the yeast (Y)
form in the animal to the mold or mycelial form(M) in the external environment
Various factors controls this YM shift (nutrients,CO2, oxidation-reduction potentials, temperature).
In plants the M form occurs in the plant and the Yform in the external environment.
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Reproduction Asexual : Fission, Budding, spore formation
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Sexual reproduction Homothalic : self-fertilizing and produce sexually
compatible gametes on the same mycelium Heterothalic: out-crossing between different but
sexually compatible mycelia
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Role of fungi in Biotechnology
Drugs (antibiotics) Food
Pesticides Pollution control Study organism ( S cerevisiae , Neurospora
crassa )
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Archaea Archaea are quite diverse group Gram positive or gram negative Shape spherical, rod-shaped, spiral, lobed,
plate-shaped, irregularly shaped, orpleomorphic.
Some are single cells, whereas others formfilaments or aggregates.
Size range from 0.1 to over 15 m Multiplication ; by binary fission, budding,
fragmentation ,
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Respiration: aerobic, facultatively, anaerobic, orstrictly anaerobic.
Energy metabolism in archaea is slightlydifferent from other bacteria.
They can be autotrophic, chemoorganotrophicor chemolithotrophic
Some are mesophiles; others arehyperthermophiles that can grow above 100C.
Some are extreme halophile, some are extremeacidophiles
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Cell structure Cell wall:
Gram + (Eg. Metanobacterium) cell wall is made upof pseudomuramic acid (N-acetyl talosamin uronicacid with (14) glycosidic bonds )
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Cell membrane:
It has a lipid monolayer made up branched chainhydrocarbons attached to glycerol by ether links
Three types of membranes are present
Bilayer of C 20diethers Monolayer of C 40 tetraethers.
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Genetic Material: Single closed DNA with ~ 56% difference with that of bacteria
and eukaryotes Classification:
The first edition of Bergeys Manual divided the archaea intofive major groups based on physiological and morphologicaldifferences
Methanogenic archaea, Archaea sulfate reducers, Extremely halophilic archaea , Cell wall less archaea , Extremely thermophilic S 0 -metabolizers
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Classification: On the basis of rRNA data archaea are devided into four
Euryarchaeota: physiologically diverse group (7 classes : Methanobacteria, Methanococci, Halobacteria,
Thermoplasmata, Thermococci, Archaeglobi, andMethanopyri)
Crenarchaeota: Mostly hyper thermophilic group Korarhcaeota: Nanoarchaeota : A parasitic prokaryote usually seen
attached to Ignicoccus a Crenarchaeota
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Oxygen classes of MicroorganismsAerobes Anaerobes
Obligate Require O 2 Micrococcusluteus
Aerotolerant Not required, butcan tolerate O
2
Streptococcus
Facultitative Not required,but grow betterwith O 2
E. Coli Obligate O 2 is harmful Methano-bacterium
Microaerophilic Required, but atlow level
Spirillumvolutans
Obligate aerobes : Aerobic respirationFacultative aerobe : Aerobic respiration, Fermentation and
Anaerobic respirationMicroaerophilic : Aerobic respiration
Obligate anaerobes : FermentationAerotolerant : Fermentation and Anaerobic respiration
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Cultivation of aerobes andanaerobes
Aerobes : agitation orbubbling to provide O 2 Anaerobes: various method
to stop O 2
Filling the bottles completelyand close with tight cap Addition of reducing agents
to convert O 2 to H 2O Thioglycolate
Bubbling the medium withN2 or H 2S
Culturing in anoxic jar orglove box
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b ( i 0 1
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Cyanobacteria (size 0.5 1m in dia. to 40 m dia.)
Photosynthetic bacteria Morphologically diverse
large group of photosynthetic bacteria
GC ratio 35 70%variation
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Different groups: Unicellular divide by binary fission (Unicellular) Unicellular divide by multiple fission forms colony
(Plurocapsalean) Non-heterocystous filaments (Oscillatorean) Filamentous with differentiated cells, heterocyst
(Nostoclean) Branching filaments (Branching)
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Structure: Peptidoglycan cellwall Many produce excessive mucilaginous envelops
Multilayered photosynthetic layers with two types of pigments : Phycobilins and Chll a Gas vesicles Some forms heterocyst with repeated cluster of nif
genes Nitrogen storage structure, cyanophycin (asp and arg) Movement by gliding
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Actimycetes is a major group in orderactinomycetales in phylum actinobacteria
Diverse, branching filamentous formingthallus
Gram + bacteria with high G+C content (
Aerobic as well as facultative aerobics Spore forming (Conidial spores) Four types of cell wall based on amino acid
or glycine in the peptidoglycan interbridge
Applications of actinomycetes ?
Actinomycetes
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Microbial Nutrition
Macro elements ( carbon, oxygen, hydrogen,nitrogen, sulfur, phosphorus, potassium,calcium, magnesium, and iron)
Micro elements (manganese, zinc, cobalt,molybdenum, nickel, and copper)
Growth factors (amino acids, purines andpyrimidines, and vitamins)
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Requirement of C H O Mostly Satisfied together Compounds act as source for C,H and O as well as
energy source Eg of Carbon sources
CO2, organic carbon sources
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Requirements for Nitrogen, Phosphorus andSulfur
Needed for the synthesis of biomolecules Nitrogen is obtained from organic (amino acids,
DNA,) or inorganic (nitrate, nitrite,) source Phosphorus is obtained from organic as well as
inorganic sources Organic P is converted to inorganic from by alkaline
phosphatase Sulfate is the preferred source of sulfur.
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Growth Factor Organic compounds required because they are
essential cell components or precursors of such
components and cannot be synthesized by theorganism are called growth factors.
Major classes of growth factors: (i) Amino acids, (ii) Purines and pyrimidines, and (iii)
vitamins.
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Uptake of Nutrients
Passive diffusion Facilitated diffusion Active transport Group translocation
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Passive diffusion: movement of molecules from aregion of higher concentration to one of lowerconcentration
The rate of passive diffusion is dependent on the
concentration gradient between a cells exterior and itsinterior A fairly large concentration gradient is required for
adequate nutrient uptake by passive diffusion , and therate of uptake decreases as more nutrient is acquiredunless it is used immediately.
Very small molecules such as H2O, O2, and CO2 oftenmove across membranes by passive diffusion
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Passive diffusion aided by a carrier proteins,(permeases), is called as facilitated diffusion.
The rate of facilitated diffusion increases withthe concentration
Each carrier is selective and will transport onlyclosely related solutes
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Eg. MIP (major intrinsic proteins) channels Aquaporins : Transport water Glycerol facilitator
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Active transport
Active transport characteristic features: Transport of solute molecules to higher
concentrations, Use of metabolic energy Involvement of carrier protein Can be inhibited by metabolic inhibitors
Eg. ATP-binding cassette transporters (ABCtransporters)
Lactase permease
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Group translocation
A process in which a molecule is transportedinto the cell while being chemically altered.
phosphoenolpyruvate: sugar phosphotransferasesystem (PTS).
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Siderophore
Siderophores are low molecular weightmolecules that are able to complex with ferriciron and supply it to the cell.
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Bacterial Growth
Increase in the number of cells in a population What is the need of studying bacterial
growth?
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DNA replication and cell elongation Formation of cell division plane Synthesis of petidoglycan Cell division
P id l h i
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Peptidoglycan synthesis Autolysin makes cut in the existing peptidogylcan
Bactoprenol transport peptidogycan precursorsthrough cytoplasmic membrane to periplasm Transpeptidation : Controlled by FtsI
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Growth Curve Lag, log and death phase
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Control of Microorganism
Sterilization: Disinfection: Pattern of Microbial death:
Exponential
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Microscopy
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Optical phenomenon used in microscopy are:reflection, diffraction and refraction
Bright field, dark field, phase contrast,fluorescence microscope.
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Resolution:
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Dark Field Microscopy
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Dark Field MicroscopyObserving live objects
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Phase Contrast Microscopy
Phase-contrast microscopy is used for Studying microbial motility Determining the shape of living cells Detecting bacterial components
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Cell division
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Generation time: time required for one cell todivide and form two cells
Binary fission During growth cycle all cellular constituents
increases proportionally and each daughtercell receives chromosomes and sufficientcopies of ribosomes and other monomers.
Fts proteins : group of proteins involved in cell
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division Divisome: division apparatus formed by Fts
proteins. Involved in peptidoglycan synthesis Synthesis of new cytoplasmic membrane
Fts Proteins
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Fts Proteins Fts (filamentous temperature-
sensitiv e) Proteins
Essential for cell division in allprokaryotes Interact to form the divisome
(cell division apparatus) FtsZ : forms ring around center of
cell ZipA: anchor that connects FtsZ
ring to cytoplasmic membrane FtsA: helps connect FtsZ ring to
membrane and also recruits otherdivisome proteins
Related to actin FtsI peptidoglycan biosynthesis
proteins FtsK separates chromosome
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DNA replicates before the FtsZ ring forms
Location of FtsZ ring is facilitated by Min
proteins MinC, MinD : Inhibits FtsZ anchoring MinE : Inhibitor of MinC and MinD present at the
center FtsK protein mediates separation of
chromosomes to daughter cells
Peptidoglycan biosynthesis and cell
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Peptidoglycan biosynthesis and celldivision
Synthesis
1. DNA replication and
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segregation2. FtsZ ring assembly along with
the formation of divisome3. Z-ring maturation4. Septal invagination with the
constriction of the envelopelayers
5. Septum closure and splittingof the daughter cells
1. FtsZ ring assembly
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1. First group of proteins assembles and anchor theFtsZ to form Z ring
2. Z ring forms as an arc at several points at themedian
3. Arc formed by the self polimerization of FtsZ
proteins with the help of GTP utilization4. FtsA and ZipA proteins anchor the micro arc on
the cell membrane
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1. Z-ring maturation after orderly recruitmentof the divisome components
1. By the ordered recruitment of late divisome
components (FtsX/E, FtsK, FtsQ, FtsN and AmiC)2. ZapA and ZapC enhance the polymerization of
the arc by aggregating the FtsZ and inhibitingGTPase activity.
3. FtsK drag Chromosome from the plane
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Septal invagination with the constriction of
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Septal invagination with the constriction of the envelope layers
Constriction is energy dependent Z-ring can exert a constrictive force onto the
membrane Simultaneously autolysin hydrolyses
peptidoglycan layer and create small opening onthe cell wall
New wall materials are added across the opening
with the help of bactoprenol and PBP
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Control of Z ring assemly
By Two complementary systems ; Min systemand Nucleoid occlusion
Min system prevents aberrant division at the
poles Fts inhibitor MinC and MinD organized to form
MinCD complex MinCD prevents the lateral assembly of FtsZ Competes with FtsA and ZipAMin E system prevents MinCD complex
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Nucleoid occlusion DNA associated proteins inhibits Z-ring formation This provides a protective mechanism to the DNA,
and contributes to the precise temporal andspatial positioning of the division septum.
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Regulation of Z ring ASSEMBLY
Growth rate dependent Z ring inhibitor
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UgtP, was identified in B. subtilis and may constitute thelink between metabolism and cell division
UgtP is an enzyme of the glucolipid biosynthesis pathwaythat uses UDP-Glucose in the synthesis of the diglucosyldiacylglycerol anchor of lipoteichoic acids
Under nutrient rich conditions, in response to high levels of its substrate, UgtP is present in the cytoplasm inhibits FtsZ
assembly, by disruption of the lateral protofilamentsinteractions during growth on a poor carbon source, the levels of UgtP
are reduced When DNA is damaged, an SOS response is activated to
repair the DNA and cease cell division by inhibiting FtsZpolymerization
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l
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DNA Replication
Initiation Elongation termination
Initiation:B i l li i i i ll d iC (245 b )
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Bacterial replication origin, called oriC (245 bp) DnaA protein bind at DnaA boxes (9mer conserved repeats) Four GATC sequences that are recognized by DNA adenine
methylase (Dam), an enzyme that methylate the adenine basewhen this sequence is unmethylated or hemimethylated Methylation of adenines alters the conformation of DNA to
promote strand separation DnaC and two DnaB proteins (helicases) bind at the 13 mer
sequence and unwind the DNA in both direction Single stranded binding proteins prevent the single strands of DNA
from forming secondary structures DNA gyrase relieve the stress created by the action of DnaB
helicase. Primase adds RNA primer to initiate DNA synthesis
l
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Elongation: Leading and lagging strand Leading strand synthesis begins with the synthesis
of a short RNA primer at the replication origin bythe enzyme Primase (DnaG protein)
Nucleotides are then added to this primer by asingle DNA polymerase III dimer . Synthesis in leading strand then proceeds
continuously, while the DNA is concurrently
unwound at the replication fork
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In lagging strand synthesis is accomplished in shortOkazaki fragments.
First, an RNA primer is synthesized by primase, DNAPol III binds to the RNA primer and addsdeoxyribonucleotides.
When the synthesis of an Okazaki fragment has beencompleted, replication halts and the core subunits of DNA Pol III dissociates
The RNA primer is remove and replaced with DNA byDNA polymerase I
The remaining nick is sealed by DNA Ligase, whichthen ligates these fragments
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Termination:
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Termination: In E.coli , there are 10 replication termini ( Ter ) located
in a region opposite to the replication origin The Ter sites interact with the replication terminator
protein called Tus, to stops DNA unwinding activity of DnaB
At the end, replication forms as catenated (twocircular chromosomes joined at ter region) ring. Catenated rings are separated by topoisomerases IV Topoisomerase IV transiently breaks both DNA strands
of one chromosome and allowing the otherchromosome to pass through the break
Plasmids
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Pl id R li ti
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Plasmid Replication
Two methods Theta formation Rolling circle replication
R lli i l li ti
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Rolling circle replication
OriRepA: Binds at the Ori, forms a nick on onestrand and remain attached to the 5 end of the strand
Free 3 end with free OH group act as theprimer for Bacterial DNA polymerase III tostart replication of plasmid.
RepA
3 OH DNA PolII
Helicase unwind the double strand,simultaneously single strand binding proteinbinds to the strand in which Rep A is attached
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Helicase
binds to the strand in which Rep A is attachedand stabilize the strand and progressivelypeeled off from the plasmid.
Once the replication of the intact strand iscomplete the RepA joins the two ends of thenicked strand.
DNA ligase seals the nick of the doublestranded DNANow the peeled single stranded DNA formsloop like structure allowing RNA polymerase tobind on it and prime the replication.
DNA polymerase starts the replication andforms dsDNA
Ss bindingprotein
Ligase
RNA poly
G T f i B t i
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Gene Transfer in Bacteria
Transformation: Uptake of free DNA Transduction: DNA transfer through a Virus Conjugation : Direct DNA transfer from one
bacteria to other
Transformation, discovered by Fred Griffith in
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Transformation, discovered by Fred Griffith in1928.
Transformation is the uptake by a cell of anaked DNA molecule or fragment from themedium and the incorporation of thismolecule into the recipient chromosome in aheritable form.
In natural transformation the DNA comes froma donor bacterium.
The process is random, and any portion of agenome may be transferred between bacteria.
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Competence: A cell that is able to take up DNA and be
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A cell that is able to take up DNA and betransformed is called as a competent cell.
Competent requires: Membrane bound DNA binding proteins Membrane bound Nucleases Competent specific proteins
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Generalized Transduction: occurs during the lyticcycle of virulent and temperate phages and cantransfer any part of the bacterial genome.
During the assembly stage, when the viral
chromosomes are packaged into protein capsids,random fragments of the partially degradedbacterial chromosome also may be packaged bymistake.
The resulting virus particle often injects the DNAinto another bacterial cell but does not initiate alytic cycle
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Specialized Transduction
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Specialized Transduction
In specialized or restricted transduction, thetransducing particle carries only specificportions of the bacterial genome
Specialized transduction is due to an error inthe lysogenic life cycle.
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