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BIOMEMBRANES AND BIOFILMS

:STRUCTURE AND FUNCTION

Presented by : Saurabh Pandey PALB-3252

Jr.M.Sc. (Agri) Plant Biotechnology

Submitted to: Dr. Dayal Doss Professor

Dept. of Plant Biotechnology

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CONTENT

BIOMEMBRANES• INTRODUCTION• STRUCTURE• FUNCTION

BIOFILMS• INTRODUCTION • STRUCTURE• INDUSTRIAL APPLICATIONS

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BIOMEMBRANES

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INTRODUCTION

Biological membranes are thin, flexible surfaces separating cells and cell compartments from their environments.

Different membranes have different properties, but all share a common architecture. Membranes are rich in phospholipids, which spontaneously form bilayer structures in water.

Membrane proteins and lipids can diffuse laterally within the membrane, giving it the properties of a fluid mosaic.

Membranes are asymmetric; interior and exterior faces carry different proteins and have different properties.

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HISTORY

1895-Charles Ernest Overton in his book on anesthesia called layers surrounding cells as ”lipoids” made of lipids and cholesterol.

1925-Gorter and Grendel proposed lipid bilayer model of cell membrane based on their experiment on RBCs extract of different animals.

1935-Danielli and Davson earliest molecular model of biomembranes including proteins with lipids.

1958-Robertsons says two protein layers are adsorbed to lipid bilayer. All membrane have same composition.

1972- The Fluid Mosaic Model of Singer and Nicolson. 1984-The Mattress Model by Mouritsen and Bloom. In 1984, Mouritsen and Bloom (1984) proposed the mattress model that

suggests that proteins and lipids display interactions with a positive free energy content due to variations in the hydrophobic length of the molecules

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Mouritsen and Bloom Mattress Model

The typical thickness of a lipid bilayer is about 5 nm.

If the hydrophobic core of a membrane protein is longer or shorter than this length, either some hydrophobic protein or lipid segments are exposed to water, or the lipid membrane has to be deformed to compensate for unfavorable hydrophobic interactions.

This effect is called as ‘hydrophobic matching’.

The hydrophobic matching give rise to interfacial tensions between lipids and proteins. These tensions may result in accumulation of certain lipids around the proteins. And in the mutual attraction of proteins due to capillary forces, leading to aggregation and clustering of proteins.

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THE MATTRESS MODEL

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S.J.SINGER AND G. NICHOLSON FLUID MOSAIC MODEL(1972)

Universally accepted model for all kinds of biological

membranes.

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BIOMEMBRANE COMPOSITION Assembly of Phospholipid bilayer and Protein embedded in it The relative amounts of protein and lipid vary significantly , ranging from about 20%

(dry wt.) protein(in myelin) to 80% protein(in mitochondria)

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Phospholipid Bilayer

Built from lipids and steroid derivatives

Phospholipids = Phosphoglycerides

Phosphoglycerides: Main ingredient.

Glycolipids

Sphingolipids

Steroids

Cholesterol

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Phospholipid structure

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Glycerol backbone connected by ester bonds to fatty acid chains

Phosphoric acid

Alcohol

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ALCOHOLS

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Give a phospholipid its name

Phosphatydylinositol

Phosphatydylcholine

Alcohol end is extremely polar (hydrophilic) Form head groups

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Decide about the properties of the phospholipid

When cleaved become important signaling molecules

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ALCOHOLS IN BIOLOGICAL MEMBRANES

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• Fatty acid end of a phospholipid molecule is strongly nonpolar (hydrophobic)

• Forms internal tails in the membrane• Usually even number of carbons• Myristate : 14• Palmitate: 16• Arachidonate: 20• Double bonds in unsaturated fatty

acid create a bend and “loosen up” membrane packing

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FATTY ACIDS

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Sphingosine (base) backbone.

Amide link to an additional fatty acid chain

Ether link to sugars or phosphoric acid and alcohol

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SPHINGOLIPIDS

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Sphingolipids

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Glycosphingolipids- Receptors for viruses

Sphingomyelins -Signaling molecules

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Another class of membrane lipids

All have four hydrocarbon rings

Cholesterol has a hydroxyl substituent on one ring

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Sterols

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Fig: Cholesterol

• Hydroxyl group can interact with water what makes the molecule amphipathic.

• Cholesterol is very abundant an necessary in of eukaryotic cells

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Cholesterols

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Fig: Phospholipid bilayer

In a bilayer Fatty acid tails point inward

Alcohol heads point outward

Each phospholipid layer is called a leaflet

Leaflets are different in composition

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Phospholipid bilayer

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How Do The Phospholipid Bilayers Form?

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Driving force are hydrophobic interactions between the fatty acid chains of phospholipids and glycolipids molecules.

Hydrogen bonds between polar groups stabilize the bilayer.

Phospholipids in biological membranes are synthesized in 2-step enzymatic reaction.

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Synthesis Of Membrane Lipids

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(A). Two fatty acids are added to glycerol 3- phosphate to produce phosphatidic acid (acyl transferases)• This steps enlarges lipid

bilayer.

(B). Phosphatase and phosphotransferase attach head groups• This steps determines the

chemical nature of lipid bilayer

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• Highly dynamic.• Lateral mobility.• Flipping between

leaflets.• Imperfectly packed fatty

acid chains (double bonds in fatty acid chains) are responsible for membrane permeability.

• High electrical resistance• Impermeable to ions• Permeable to gases and small

lipid solublemolecules• Slightly permeable to water• Ability to self seal (always form

closedcompartments)

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Physical properties of the phospholipid bilayer

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MEMBRANE PROTEINS

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• Each cell membrane has a set of specific membrane protein .

• Membrane proteins allow the membrane to carry out its distinctive functions

• Membrane proteins are either integral (intrinsic) or peripheral

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• Cross the bilayer (transmembrane)

• Transmembrane segment is usually α helix

• A segment of 25 hydrophobic residues

• Examples: G protein coupled receptors, ion

channels, pumps, transporters

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Integral (intrinsic) membrane Proteins

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(A) Glycophorin• Single transmembrane domain protein

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Examples of intrinsic membrane proteins

(B) Bacteriorhodopsin• Multiple transmembrane domains protein

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• Do not interact with hydrophobic core of the bilayer

• Are associated with membrane through lipid anchors

• Interactions with bilayer (but not complete crossing) or contact with integral membrane proteins

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Peripheral membrane proteins

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• Transport of nutrients• Passage of water• Selective transport of molecules

(keep the unwanted molecules out, secrete metabolic by products)

• Maintenance of proper ionic composition inside the cell

• Reception of signals from the extracellular environment

• Expression of cell identity• Physical and functional

connection with other cells or the extracellular matrix (in multicellular organisms)

Functions of membrane proteins

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Asymmetry Of The Membrane

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The two faces of a membrane are asymmetrical in lipid and protein composition

All integral and membrane bound proteins are distributed asymmetrically

Each protein has a single, specific orientation with respect to cytosolic and exoplasmic faces of the membrane

Glycolipids are located exclusively on the exoplasmic leaflet

3027/11/13Fig:Asymmetry Of The Membrane

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Protein: lipid ratio in the membrane depends on the function

Mitochondrial membrane is 76% protein (has many transporters and enzymes). Also in purple membrane of halobacteria

Myelin (Schwann cell) membrane has 18% protein (phospholipids are great insulators)

Fig: Light harvesting complex of purple bacteria

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Plasma membranes have different protein:lipid ratio

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FUNCTIONS

SELECTIVE PERMEABILITY

PINOCYTOSIS

CELL RECOGNITION

BIOGENESIS OF CELL ORGANELLES

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA MEMRANE

ABSORPTION AND SECRETION IN INTESTINAL CELLS

AS A HORMONE RECEPTOR SITES

CELL ADHESION

TRANSMISSION OF NERVE IMPULSE

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MEMBRANE PERMEABILITY

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MEMBRANE TRANSPORT

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CELL RECOGNITION

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THE WATER CHANNEL

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Discovery of these water channels led to a Nobel Prize in Chemistry in 1993 to Dr. Peter Agre

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PINOCYTOSIS

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NANOTECHNOLOGY IN BIOMEMBRANES

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WHAT ARE BIOFILMS

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• Biofilms are colonies of living micro-organisms (e.g., bacteria, fungi, algae, and/or protozoa) growing on any surface (e.g. metals, plastics, tissue, soil particles, teeth, and so forth)

• Biofilms are surface-attached communities of bacteria, encased in an extracellular matrix of secreted proteins, carbohydrates, and/or DNA, that assume phenotypes distinct from those of planktonic cells

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HISTORY

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In 1684 Anthony van Leeuwenhoek remarked on the vast accumulation of microorganisms in dental plaque in a report to the Royal Society of London: "The number of these animicules in the scurf of a man's teeth are so many that I believe they exceed the number of men in a kingdom

In a 1940 issue of the Journal of Bacteriology, authors H. Heukelekian and A. Heller wrote, "Surfaces enable bacteria to develop in substrates otherwise too dilute for growth. Development takes place either as bacterial slime or colonial growth attached to surfaces." Claude ZoBell described many of the fundamental characteristics of attached microbial communities in the 1940s

The earliest use of “biofilm” in publication is in the Swedish journal Vatten: Harremoës, P. 1977. “Half-order reactions in biofilm and filter kinetics,” Vatten, 33 122-143

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CONTINUED...

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The earliest use of “biofilm” in publication is in the Swedish journal Vatten: Harremoës, P. 1977. “Half-order reactions in biofilm and filter kinetics,” Vatten, 33 122-143

In 1990, recognizing the significance of microbial activity, as well as the tremendous economic costs associated with microbial communities on surfaces, the US National Science Foundation founded the Center for Biofilm Engineering at Montana State University in Bozeman (though, interestingly, NSF would not initially accept the word “biofilm” in the Center’s name; instead the award funded the “Center for Interfacial Microbial Process Engineering”)

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FORMATION OF BIOFILMS

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Form in places with access to water

Attach to a solid surface using several means of Flagella, Hydrophobic Cell Walls & Sticky Polymers

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STEPS IN BIOFILM FORMATION

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• Structure of biofilms are dramatically different due to the specific organisms in the film and environmental conditions

Interaction of cells with a surface or with each other(A) •Initiation of biofilm formation

(B) •Films aggregate

(C) •Cells form an extracellular matrix

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STEPS OF BIOFILM DEVELOPMENT

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STRUCTURE OF BIOFILMS

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Key components of the Biofilm matrix are extracellular polysaccharides and proteins

Dead cells have also been identified in biofilms

Extracellular DNA is also important

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Polysaccharides in Biofilms

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Carbohydrates significantly impact bacterial virulence

Bacteria have capsular polysaccharides and exopolysaccharides

The polysaccharides are not soluble and do not disassociate with the bacterial cells

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Proteins in biofilms

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The biofilm associated protein (BAP)Structurally similiar to the surface proteins

Esp of Enterococcus faecalismus20 of Pseudomonas aeruginosasty2875 of Salmonella typhi

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PATHOGENS THAT HAVE BEEN STUDIED FOR THE FORMATION OF BIOFILMS

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Staphylococcus aureus- for urinary catheters in medical industry

Staphylococcus mutans-In human dental caries

Salmonella typhi-For microbial cantamination of food in food industry

Enterococcus faecalis-Endocarditis and biofilm associated pili

Pseudomonas aeruginosa- tobramycin resistance and growing on urinary catheters

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GENES AND BIOFILMS

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In November 2005,Biologist Alejandro Toledo Arana has identified two genes(arlRS,sarA) that regulate the formation of biofilms in Staphylococcus aureus

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BIOFILMS CHARACTERISTICS

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Submerged biofilms seems to form columns and mushroom like projections that are separated by water-filled channels

Floating biofilms form a skin or pellicle at the air- liquid interface – shows organization of cells with the matrix at the outside

Films that form on the surface of solid media such as agar or other surfaces

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CONTINUED……

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Top to bottom gradient of decreasing antibiotic susceptibility

The gradient originates in the surface layers of the biofilms where there is complete consumption of oxygen and glucose

There are patches of antibiotic resistance at the surface

Proximity of cells lead to horizontal transfer of genes for resistance

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BIOFILMS –QUORUM SENSING

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Certain species of bacteria communicate with each other within the biofilm. As their density increases, the organisms secrete low molecular weight molecules that signal when the population has reached a critical threshold. This process, called quorum sensing, is responsible for the expression of virulence factors.

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USES OF BIOFILMS

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Often used to purify water in water treatment plants

Used to break down toxic chemicals

Used to produce useful biological compounds, including medicines

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PROBLEMS CAUSED BY BIOFILMS

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Tend to clog pipes and water filters

Can cause

numerous diseases

, including many diseases prevalent in hospitals

Extr

a-resistant to antibiotics

Can

form al

most

anywhere tha

t water is

present,

including catheters, kitchen

counters, etc

.

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AGENTS FOR DESTRUCTION OF BIOFILMS (INDUSTRIAL BIOCIDES)

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(Alexidine, Chlorhexidine, Polyhexamethylene biguanides), monophenylethers (Phenoxyethanol) and quaternary amonium compounds (Cetrimide, Benzalkoniums) and have demonstrated biochemical bases for the activities and associated mammalian cell toxicities of thiol interactive agents (bronopol, isothiazolones).

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INDUSTRIAL APPLICATIONS

Bioremedation-Bacterial degradation of polluting environments.(Pseudomonas aeruginosa)Biofilteration-Selective removal of chemicals in solution. Use of Moving Bed Biofilm Reactor Technology.Biobarriers-Protection of objects using extremely rugged glycocalyx produced by biofilms.(Grodonia polyisoprenivorens)Bioreactors-Production of compounds using engineered biofilms.

BIOFILMS IN MEDICAL DEVICES-

•Contact lenses•Central venous catheters•Endotracheal tubes•Intrauterine devices•Mechanical heart valves•Pacemakers•Dialysis catheters•Urinary catheters•Voice prostheses

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MEDICAL APPLICATION

FIG:CENTRAL VENOUS CATHETORS

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MOVING BED BIOFILM REACTOR

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MBBR

TRICKLING FILTER

ACTIVATED SLUDGE

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REFERENCES

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doi: 10.1128/AAC.27.4.619Antimicrob. Agents Chemother.April 1985 vol. 27 no. 4 619-624

Research Article International Food Research Journal 18: 31-38 (2011)

J Clin Invest. 2006;116(10):2799–2807. doi:10.1172/JCI29021. Copyright © 2006, American Society for Clinical Investigation

doi: 10.1128/AEM.71.5.2372-2380.2005Appl. Environ. Microbiol. May 2005 vol. 71 no. 5 2372-2380

doi: 10.1128/JB.187.15.5318-5329.2005J. Bacteriol. August 2005 vol. 187 no. 15 5318-5329

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THANK YOU