Microbiology Basic and Applied

7/27/2019 Microbiology Basic and Applied

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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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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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Microbiology Basic and Applied