BACTERIAL CYTOLOGY

CELL WALLWHY STUDY-Determines shapeProtects cellProtection from toxic substancesSite of action for antibiotics

LOCATION-Outermost, rigid layer

TYPES-based on Gram staining

• Gram Positive, purple colour• Gram Negative, pink colour (Christian Gram-1884)

Structural differences between G+ve & G-ve cell wall

• 20-80% nm thick peptidoglycan (murein)

• 2-7% nm thick peptidoglycan

PEPTIDOGLYCAN STRUCTURE- G+ve1. Amino sugars N acetyl glucosamine & N acetyl muramic acid(NAG & NAM)2. Protein3. Techoic acid

TECHOIC ACID

Mesh structure of peptidoglycan

GRAM NEGATIVE CELL WALL

BLP( Braun’s lipoprotein)• Braun's lipoprotein found in some gram-negative cell

walls• the most abundant membrane proteins• It is bound by a covalent bond to the peptidoglycan layer• and is embedded in the outer membrane by its

hydrophobic head • BLP tightly links the two layers and provides structural

integrity to the outer membrane.

• Braun's Lipoprotein consists of phospholipids and Lipopolysaccharide.

Gram Negative Cell Wall-chemical compostion

• Lipopolysaccharide (LPS)Large, complex molecule with lipid & carbohydrate1. Lipid A2. Core polysaccharide3. O side chains

Functions of LPS• Give negative charge to the surface• Helps in attachment• Stabilize the membrane• Create a permeability membrane• Prevent entry of toxic substances• O side chain protects bacteria• Lipid A is toxic (endotoxin)- causes serious

septic condition in the body

Functions of LPS• Outer membrane is more permeable

than pm due to porins• Porins are proteinic in nature, tube

shaped, allows passage of molecules smaller than 600-700d• For larger molecules carrier proteins

are there

Periplasmic space in bacteria

Periplasmic space in bacteria• Gram Positive• Small• Fewer proteins• Proteins present,

attached to plasma membrane

• Exoenzymes- degrade polymeric nutrient

• Gram Negative• Wide (30nm-70nm)• More proteins• Hydrolytic enzymes,

transport proteins• Electron transport

proteins• Proteins for ppd syn• Modify toxic compd

COMPONENTS EXTERNAL TO CELL WALL

Capsule, Slime layer and S layerCapsule- chemical structure-1. Polysaccharide2. Protein3. Polysaccharide-Protein

GLYCOCALYX(capsule,slime)

• When the layer is well organized and not easily washed out

• When it is a zone of diffuse, unorganized material that is removed easily

Capsule under the microscope

Functions of Capsule

• Resist phagocytosis

• Storing water, prevents from desiccation

• Helps in attachment

S layer

• External to cell wall

Functions of S layer1. Protection against -• ions and pH fluctuation• Adverse surroundings2. Maintains shape3. Promotes adhesion4. Adds the property of virulence

Pilus (pili, fimbriae)• Short, fine, hair like appendages• Visible under electron microscope

only• One cell may have 1000 of pili• Slender tube • 3-10nm in diameter, several

micrometer in length

PILI

PILI

Pilus

Helically arranged pilin proteins

Chemical composition-Protein- PILIN

Functions of Pili• Attachment-rock surface, host cell

• Some may help in motility eg type IV- jerky motility up to several mm

• Gliding motility eg. Myxobacteria

• conjugation

Flagella(flagellum)• Thread like locomotory organelle• Extending outward from the cw and pm• Slender, rigid structure• 20nm across and 15-20micrometer long• Stained and can be seen under

compound ms• Ultrastructure under electron ms

Arrangement of flagella

Ultrastructure of flagella

Under TEM

Ultra structure of flagella

Flagellar ultrastructure

Three parts1. Filament2. Basal body3. hook

Ribosome

Ribosomes Location- cytoplasm and some attached to pmcomplex structureComposition- protein and ribonucleic acid (RNA)Parts- 50s and 30s(s- svedberg unit)Function- protein synthesisFolding of protein- by special protein called chaperone

Ribosome contd.

Size- 14-15nm by 20nmMol wt.-2.7 million

The nucleoid

The most striking feature

no nuclear membraneLocated in an irregularly shaped

region called nucleoid

Other namesNuclear body, chromatin, nuclear

region

Forms

1. Double stranded DNA(deoxyribonucleic acid)2. Linear chromosome3. Some have more than one chromosome

• A chromosome is a structure of DNA, protein, and RNA found in cells.

• It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences.

• Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.

• DNA encodes most or all of an organism's genetic information;

• some species also contain plasmids or other extra chromosomal genetic elements.

Electron micrograph

Chemical analysis• 60% DNA• 30% RNA• 10% Protein• E.coli-DNA circle-1400µm230-700 times longer than the cellLooped and coiled efficientlyNo histone protein

Exceptions- Perillulla & Gemmata

Gemmata

plasmid

Extra chromosomal DNA material

PlasmidExamples- bacteria, fungi, yeast.Small, double stranded DNA moleculeExist independent of chromosomeLinear and circularFew genes –less than 30Not essential for survivalSelective advantage

PlasmidReplicate autonomouslySingle copy produces one copy per cellAble to integrate into the chromosome and gets replicated – episomeSometimes lostThe loss of plasmid- curing

Curing agents• UV and ionizing radiation• Thymine starvation• Antibiotics• Growth above optimal

temperature

Types of Plasmidstype host function

Fertilty factor E.coli conjugationMetabolic plasmids

E.ColiRhizobium

Lactose degradation& symbiosisNitrogen fixation

R plasmid Pseudomonas Resistance to antibiotic

Col plasmid E.coli Colicin production

Virulence plasmid

E.coli Entrotoxin, siderophore

Cell membrane• Retains cytoplasm• Selective membrane• Prevents loss of essential

components• Transport system•Waste excretion• Protein secretion

Plasma membrane-functions

• Location for respiration, photosynthesis, synthesis of lipid and cell wall constituents• Has receptor molecule to detect and

respond to chemicals in the surrounding• Essential for the survival of bacteria

Fluid Mosaic Model

Fluid Mosaic Model of Singer & Nicolson

• Bilayer phospholipid (amphipathic)• Proteins float within• 5-10 nm thickness• Polar hydrophilic head• Long non polar hydrophobic end• Proteins-peripheral-20-30%,integral-60-80%• No cholesterol but hopanoids

Internal membraneTubule ,vesicle, lamellae

Cyanobacteria-infoldings are complexSpherical & flattened vesicle, tubular membranes

Inclusion structuresCarbon storage polymers

1. Poly-β-hydroxybutyric acid

1. PHB

Length:C3-C18

PHBSynthesis when carbon is in excess and used for biosynthesis and to make ATPPHB are referred to as poly-β-hydoxyalkonate(PHA)2. glycogen- polymer of glucoseStore house of carbon and energy

Polyphosphates

Polyphosphate contd

Functions- source of phosphateUsed as sources of phosphates for nucleic acid and phospholipid biosynthesis Note- phosphate in the environment is limited

Sulfur Granules

Elemental sulfur accumulated inside the cell

Sulfur

• Oxidize reduced sulfur(H2S)

Magnetosomes

Magnetosomes

• Some bacteria can orient themselves within magnetic field because of magnetosomes- magnetotaxis

• These are intracellular particles• Iron materials• Impart magnetic dipole on a cellFunctions-not knownMay be guiding bacteria towards magnetic field deep in aquatic envt. Where oxygen level is low

Magnetosomes• Surrounded by a membrane

containing phospholipid, proteins, and glycoprotein• Proteins act as chelating agents• Square to rectangular in shape to

spike shaped

Gas vesicle• Planktonic- those live in floating state• Because of gas vesicles• These structure confer buoyancy on cell• Eg. Cyanobacteria also called

BGA(bloom)• Purple and green sulfur bacteria• ArchaeaNote- Eucaryotes don’t have these structure

bloom

bloom

General structure• Spindle shaped filled with gas• Made up of protein• Hollow yet rigid• Variable length and diameter• 300nm-1000nm by 45nm-120nm• Number-few to 100 per cell• Membrane made up of protein, 2mm thick• Impermeable to water and solute but

permeable to gas

Gas vesicle contd.• Clusters of vesicles- gas vacuole• Can be seen under light microscope and TEM

Molecular structure• Major gas vesicle protein-GvpA- small,

hydrophobic and rigid(97%)• Minor protein-GvpC

Other inclusion

• Cynophysin and granules- equal amount of arginine and aspartic acid

• Store extra amount of nitrogen

Carboxysome present in photosynthetic bacteriaPolyhedral, 100nm in diameter, contains enzyme ribulose 1, 5 biphosphate carboxylase(Rubisco)

Endospore

Endospore• Endo-within• Enable cells to endure difficult time-Temperatures, drying, nutrient depletion etc.Dormant stage of bacteria-Used for dispersalExamples-Bacillus, Clostridium

Electron micrograph of endospore

Schematic presentation of endospore

Endospore formation and germination

Activation- Use of elevated temperatureGermination-Specific nutrient like alanineOutgrowth

Three stagesActivation- endospores get ready for germinationGermination- rapid process, spore loses its refractibilityOutgrowth-swelling due water up take, synthesis of new RNA, proteins &DNA.

characteristics• Dipicolinic acid in core (with calcium)in

core (10%)- reduces the water content of core.• Endospores become dehydrated1. Increases heat resistance2. Makes cells resistant to chemicals3. Keeping enzymes inactive in the core

characteristics• pH one unit lower than vegetative cell• High level of SASPs(small acid soluble

proteins)1. Bind tightly to DNA in the core –protection

from UV, desiccation, and dry heat(DNA changed from B form to the compact form A which is more resistant to mutation, denaturing effect of dry heat)2. Carbon and energy source

Difference between vegetative cell & endospore

Vegetative cell Endospore