Microbiology Basic and Applied

Dr. Bipinraj N K

Role of Microbiology
Medical Industrial Molecular Biology Environmental Genetics & Recombinant DNA Tech

Microorganisms or Microbes
Microscopic organism Bacteria Fungi Algae Protozoa Virus

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

Bacterial Cell Wall :GTeichoic + acids, polymers o

It has negative charge 80 nm

glycerol or ribitol joined by phosphate group

60 nm

Bacterial Cell Wall : G -

8 nm

Fungi
Eukaryotic organism Unicellular (yeast) and multicellular (mushrooms) Chitin in the cellwall Heterotrophic & no chloroplast Reproduction by sexual & asexual

 Cell structure
Single cellualr: yeast Multicellualr : mold
Long, branched, threadlike filaments of cells called hyphae
Septate Ceonocytic

 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 Y form in the external environment.

 Reproduction
Asexual : Fission, Budding, spore formation

 Sexual reproduction
Homothalic : self-fertilizing and produce sexually compatible gametes on the same mycelium Heterothalic: out-crossing between different but sexually compatible mycelia

Role of fungi in Biotechnology

Drugs (antibiotics) Food Pesticides Pollution control Study organism (S cerevisiae, Neurospora crassa )

Archaea
Archaea are quite diverse group Gram positive or gram negative Shape spherical, rod-shaped, spiral, lobed, plate-shaped, irregularly shaped, or pleomorphic. Some are single cells, whereas others form filaments or aggregates. Size range from 0.1 to over 15m Multiplication ; by binary fission, budding, fragmentation,

 Respiration: aerobic, facultatively, anaerobic, or strictly anaerobic. Energy metabolism in archaea is slightly different from other bacteria. They can be autotrophic, chemoorganotrophic or chemolithotrophic Some are mesophiles; others are hyperthermophiles that can grow above 100C. Some are extreme halophile, some are extreme acidophiles

 Cell structure
Cell wall:
Gram + (Eg. Metanobacterium) cell wall is made up of pseudomuramic acid (N-acetyl talosamin uronic acid with (14) glycosidic bonds )

 Cell membrane:
It has a lipid monolayer made up branched chain hydrocarbons attached to glycerol by ether links Three types of membranes are present

Bilayer of C20diethers

Monolayer of C40tetraethers.

 Genetic Material:
Single closed DNA with ~ 56% difference with that of bacteria and eukaryotes

Classification:
The first edition of Bergeys Manual divided the archaea into five major groups based on physiological and morphological differences

Methanogenic archaea, Archaea sulfate reducers, Extremely halophilic archaea , Cell wallless archaea ,

Extremely thermophilic S0 -metabolizers

 Classification:
On the basis of rRNA data archaea are devided into four

Euryarchaeota: physiologically diverse group

(7 classes : Methanobacteria, Methanococci, Halobacteria, Thermoplasmata, Thermococci, Archaeglobi, and Methanopyri)

Crenarchaeota: Mostly hyper thermophilic group Korarhcaeota: Nanoarchaeota : A parasitic prokaryote usually seen attached to Ignicoccus a Crenarchaeota

Oxygen classes of Microorganisms

Aerobes
Obligate Facultitative Require O2 Not required, but grow better with O2 Required, but at low level Micrococcus luteus E. Coli Aerotolerant Obligate

Anaerobes
Not required, but can tolerate O2 O2 is harmful Streptococcus Methanobacterium

Microaerophilic

Spirillum volutans

Obligate aerobes Facultative aerobe Microaerophilic Obligate anaerobes Aerotolerant

: : : : :

Aerobic respiration Aerobic respiration, Fermentation and Anaerobic respiration Aerobic respiration Fermentation Fermentation and Anaerobic respiration

 Cultivation of aerobes and anaerobes

Aerobes : agitation or bubbling to provide O2 Anaerobes: various method to stop O2
Filling the bottles completely and close with tight cap Addition of reducing agents to convert O2 to H2O
Thioglycolate

Bubbling the medium with N2 or H2S Culturing in anoxic jar or glove box

Cyanobacteria (size 0.5 1 m in dia. to 40 m dia.)

Photosynthetic bacteria Morphologically diverse large group of photosynthetic bacteria GC ratio 35 70% variation

 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)

 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

Actinomycetes
Actimycetes is a major group in order actinomycetales in phylum actinobacteria Diverse, branching filamentous forming thallus 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 ?

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 and pyrimidines, and vitamins)

 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

 Requirements for Nitrogen, Phosphorus and Sulfur

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.

 Growth Factor
Organic compounds required because they are essential cell components or precursors of such components and cannot be synthesized by the organism are called growth factors. Major classes of growth factors:
(i) Amino acids, (ii) Purines and pyrimidines, and (iii) vitamins.

Uptake of Nutrients
Passive diffusion Facilitated diffusion Active transport Group translocation

 Passive diffusion: movement of molecules from a region of higher concentration to one of lower concentration The rate of passive diffusion is dependent on the concentration gradient between a cells exterior and its interior A fairly large concentration gradient is required for adequate nutrient uptake by passive diffusion , and the rate of uptake decreases as more nutrient is acquired unless it is used immediately. Very small molecules such as H2O, O2, and CO2 often move across membranes by passive diffusion

 Passive diffusion aided by a carrier proteins, (permeases), is called as facilitated diffusion. The rate of facilitated diffusion increases with the concentration Each carrier is selective and will transport only closely related solutes

 Eg. MIP (major intrinsic proteins) channels

Aquaporins : Transport water Glycerol facilitator

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 (ABC transporters) Lactase permease

Group translocation
A process in which a molecule is transported into the cell while being chemically altered.
phosphoenolpyruvate: sugar phosphotransferase system (PTS).

Siderophore
Siderophores are low molecular weight molecules that are able to complex with ferric iron and supply it to the cell.

Bacterial Growth
Increase in the number of cells in a population What is the need of studying bacterial growth?

DNA replication and cell elongation Formation of cell division plane Synthesis of petidoglycan Cell division

 Peptidoglycan synthesis
Autolysin makes cut in the existing peptidogylcan Bactoprenol transport peptidogycan precursors through cytoplasmic membrane to periplasm Transpeptidation : Controlled by FtsI

 Growth Curve
Lag, log and death phase

Control of Microorganism
Sterilization: Disinfection: Pattern of Microbial death:
Exponential

 Factors controlling effectiveness of antimicrobial effectiveness

Population size Population composition Concentration or intensity of an antimicrobial agent Duration of exposure Temperature Local environment
pH Organic compounds present

Microscopy

 Optical phenomenon used in microscopy are: reflection, diffraction and refraction Bright field, dark field, phase contrast, fluorescence microscope.

Resolution:

Dark Field Microscopy

Observing live objects

Phase Contrast Microscopy

Phase-contrast microscopy is used for
Studying microbial motility Determining the shape of living cells Detecting bacterial components

Cell division

 Generation time: time required for one cell to divide and form two cells Binary fission During growth cycle all cellular constituents increases proportionally and each daughter cell receives chromosomes and sufficient copies of ribosomes and other monomers.

 Fts proteins : group of proteins involved in cell division Divisome: division apparatus formed by Fts proteins.
Involved in peptidoglycan synthesis Synthesis of new cytoplasmic membrane

Fts Proteins

Fts (filamentous temperaturesensitive) Proteins

Essential for cell division in all prokaryotes 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 other divisome proteins Related to actin FtsI peptidoglycan biosynthesis proteins FtsK separates chromosome

 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 division

Synthesis

1. DNA replication and segregation 2. FtsZ ring assembly along with the formation of divisome 3. Z-ring maturation 4. Septal invagination with the constriction of the envelope layers 5. Septum closure and splitting of the daughter cells

1. FtsZ ring assembly

1. First group of proteins assembles and anchor the FtsZ to form Z ring 2. Z ring forms as an arc at several points at the median 3. Arc formed by the self polimerization of FtsZ proteins with the help of GTP utilization 4. FtsA and ZipA proteins anchor the micro arc on the cell membrane

1. Z-ring maturation after orderly recruitment of 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 inhibiting GTPase activity. 3. FtsK drag Chromosome from the plane

 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 on the cell wall New wall materials are added across the opening with the help of bactoprenol and PBP

Control of Z ring assemly

By Two complementary systems ; Min system and 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 ZipA Min E system prevents MinCD complex

 Nucleoid occlusion
DNA associated proteins inhibits Z-ring formation This provides a protective mechanism to the DNA, and contributes to the precise temporal and spatial positioning of the division septum.

Regulation of Z ring ASSEMBLY

 Growth rate dependent Z ring inhibitor

UgtP, was identified in B. subtilis and may constitute the link between metabolism and cell division UgtP is an enzyme of the glucolipid biosynthesis pathway that uses UDP-Glucose in the synthesis of the diglucosyl diacylglycerol 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 protofilaments interactions 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 FtsZ polymerization

DNA Replication
Initiation Elongation termination

 Initiation:
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 base when 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

 Elongation:
Leading and lagging strand Leading strand synthesis begins with the synthesis of a short RNA primer at the replication origin by the enzyme Primase (DnaG protein) Nucleotides are then added to this primer by a single DNA polymerase III dimer. Synthesis in leading strand then proceeds continuously, while the DNA is concurrently unwound at the replication fork

 In lagging strand synthesis is accomplished in short Okazaki fragments. First, an RNA primer is synthesized by primase, DNA Pol III binds to the RNA primer and adds deoxyribonucleotides. When the synthesis of an Okazaki fragment has been completed, replication halts and the core subunits of DNA Pol III dissociates The RNA primer is remove and replaced with DNA by DNA polymerase I The remaining nick is sealed by DNA Ligase, which then ligates these fragments

 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 (two circular 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 other chromosome to pass through the break

Plasmids

Plasmid Replication
Two methods
Theta formation Rolling circle replication

Rolling circle replication

Ori

RepA: Binds at the Ori, forms a nick on one strand and remain attached to the 5 end of the strand Free 3 end with free OH group act as the primer for Bacterial DNA polymerase III to start replication of plasmid.
DNA PolII

RepA

3 OH

Helicase

Helicase unwind the double strand, simultaneously single strand binding protein binds to the strand in which Rep A is attached and stabilize the strand and progressively peeled off from the plasmid. Once the replication of the intact strand is complete the RepA joins the two ends of the nicked strand.

Ss binding protein

DNA ligase seals the nick of the double stranded DNA Now the peeled single stranded DNA forms loop like structure allowing RNA polymerase to bind on it and prime the replication. DNA polymerase starts the replication and forms dsDNA

RNA poly Ligase

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 1928. Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule into the recipient chromosome in a heritable form. In natural transformation the DNA comes from a donor bacterium. The process is random, and any portion of a genome may be transferred between bacteria.

 Competence:
A cell that is able to take up DNA and be transformed is called as a competent cell. Competent requires:
Membrane bound DNA binding proteins Membrane bound Nucleases Competent specific proteins

Transduction
Transduction is the transfer of bacterial genes by viruses.
Generalized Transduction Specialized Transduction

 Generalized Transduction: occurs during the lytic cycle of virulent and temperate phages and can transfer any part of the bacterial genome. During the assembly stage, when the viral chromosomes are packaged into protein capsids, random fragments of the partially degraded bacterial chromosome also may be packaged by mistake. The resulting virus particle often injects the DNA into another bacterial cell but does not initiate a lytic cycle

Specialized Transduction
In specialized or restricted transduction, the transducing particle carries only specific portions of the bacterial genome Specialized transduction is due to an error in the lysogenic life cycle.