Chapter 4

Bacterial Structure and Growth

Bacteria

• Bacteria can be classified by their morphology (shape), stain reactions, atmospheric requirements, growth needs, organization, physical and biochemical composition and antigenic structure.

MorphologyShapes and Arrangements (Pg 122 – 124)

• Three common shapes– Bacilli (rod)

• Long as 20 µm or as short as 0.5 µm

– Cocci (sphere)• Kokkos meaning “berry”• 0.5 to 1.0 µm

– Spirochetes (spiral)• 1 to 100 µm in length

Structure of Bacteria Pg. 122 to 139

Flagella

• Motility – is the independent movement of an organism.

• In bacteria movement is carried out by flagella.

• Each flagella is composed of a filament, hook and basal body.

Flagella

• The long filament is composed of a helical protein strand.

• Subunits of the protein filaments are called flagellin.

• The filament is anchored into the cell membrane by hook which is attached to the basal body.

• Basal body is made up of 20 protein that forms a central rod and closing rings.

• There can be two to four rings making up the basal body.

• Range in length 10 to 20 um and 20nm thick– To view need to stain

Arrangement of Flagella

• Monotrichous – single flagellum at one end.

• Lophotrichous – two or more flagella at one end.

• Amphitrichous – single or group of flagella at both ends.

• Peritrichous – flagella over the whole cell surface.

Movement

• Flagella spin counter-clockwise to move the bacteria forward.

• The flagella reverse (Clock-wise) to move the bacteria back.

• The backward movement is more like a tumble that helps change direction not backup the bacteria.

• Most movement is stimulated by chemicals and light and is known as taxis – Light taxis– Chemotaxis

Pili (Fimbriae) • Structures used for attachment to specific surfaces.

• Many Gram negative bacteria have pili.

• Short hair like fibers.

• Made up of protein subunits called pilin.

• Have protein adhesion located at the tip of the pili that bind to different animal tissue.

• Neisseria gonorrhoeae pili attach to urogenital tract (or toilet seat).

Conjugation Pili

• True pili is used in conjugation (transfer of DNA from one bacteria to another).

• It is a tube that attaches to a bacteria.

• The DNA is moved down the tube from the donor to the receiver.

Capsule (Glycocalyx)• Many bacteria have capsules.

• Bacteria will secrete layer of polysaccharides and proteins.

• The capsule serves as a buffer between the bacteria and the environment to prevent dehydration.

• It also helps to attach the cell to each other and to host (V. cholerae to intestinal wall).

• Protects some bacteria from being engulfed by white blood cells.

Cell Wall• All bacteria except for Mycoplasma have a cell wall.

• A cell wall protects the bacteria and gives it its shape.

• Major role is to prevent lysis (bursting) due to osmotic pressure.

• Cells have more dissolved material inside them then is found in the surrounding environment.

• This hypertonic condition would cause water to move into the cell an increase the pressure inside.

• Without the cell wall the bacteria would burst.

Cell Wall• Eubacteria cell walls are made up of a network

of peptidoglycan chains.

• Peptidoglycan is made up of alternating units of two amino containing sugars.– N-acetylglucosamine (NAG)– N-acetylmuramic acid (NAM)

• This carbohydrate backbone is held together by side chains of four amino acids.

• Plus polypeptide cross-linkage

Cell Wall Gram-Positive Bacteria

• Gram-positive bacteria have thick layered peptidoglycan wall.

• 60 to 90 % made up of peptidoglycan.

• Peptidoglycan layer also has a anionic polysaccharide called teichoic acid.

• Helps to link peptidoglycan layers together.

• Penicillin interferes with the construction of the peptidoglycan layer.

Insert Picture of Peptidoglycan

Cell Wall of Gram-Negative Bacteria

• Gram-negative bacteria have a multilayered cell wall.

• No teichoic acid.

• Peptidoglycan layer much thinner.

• Peptidoglycan makes only 10% total of cell wall.

Gram-negative Cell Wall Layers• Outer membrane

– Made up of bilayer.

– Inner layer of phospholipids.

– Outer layer of lipopolysaccharides (LPS).

– Contains proteins porins • Porins form pores in the outer membrane.

• Allows small molecules to move through the cell wall.

• Inner layer is made up of peptidoglycan.

Gram-Negative Cell Wall LPS

• Lipopolysaccharide (LPS) are not found in any other organism.

• The lipid portion (lipid A) is found in the outer membrane bi-layer.

• Lipid A portion is a endotoxin– When the bacteria die they breakdown and the LPS is

released and causes fever and circulatory collapse.

• The polysaccharide is a O-polysaccharide – Used to identify bacteria ie O157:H7

Bacterial Cell Membrane• Boundary between cytoplasm and external

environment.

• Act as a permeability barrier to hold bacterial cytoplasm inside the cell.

• Also acts to transport nutrients in and waste out of the cell.

• Made up of 40% phospholipids and 60% proteins.

• Phospholipids are arranged in two parallel layers (bi-layer).

Cell Membrane

• Proteins in the membrane can be used for synthesis of the membrane.

• Also, some of these proteins are used for energy production (ATP).

• Other proteins act as transporters of charged molecules in and out of the bacteria.

Cytoplasm (Pg 135 to 136)

• Cytosol a gelatinous mass of proteins, amino acids, sugars, nucleotides, salts, vitamins and ions.

• Structures– Ribosomes hundreds of thousands in bacteria– Made of RNA and protein– Used for protein synthesis

Cytoplasm • Inclusion bodies

– Store nutrients or monomers for bacterial structures– Metachromatic granules – phosphate depots– Magnetosome crystals of iron-containing molecules

magnetite.• Help align bacteria to magnetic fields

• Nucleoid – chromosome region– No membrane DNA aggregates and no ribosomes

are found in that area.

• Plasmids – small circular DNA– Can be one or more– 5-100 genes– Important function in causing disease

DNA Plasmids

• Some plasmids carry genes for drug resistance.

• R-plasmids (R = “resistance”).

• These plasmids can be transferred between bacteria during conjugation.

Endospores• A few Gram-positive bacteria produce a highly

resistant structures called endospores (spores).

• Bacillus and Clostridium Genuses are of major concern in human bacterial infections and are spore produces.

• Endospores will be produced by these bacteria due to nutritional or environmental pressures.

• Endospores are one of the most resistant living things known.

Endospores

• Little water and undergo few chemical reactions.

• Have some ribosomes and enzymes.

• Has an unique organic substance called dipicolinic acid.

• Dipicolinic acid helps stabilize proteins and DNA.

• Helps to make spores viable at 100ºC for 2 hr.

Endospore

Diseases Caused by Spore Forming Bacteria

• Anthrax caused by Bacillus anthracis.

• Botulism, gas gangrene and tetanus caused by different Clostridium species.

• Clostridium difficile when you take antibiotics this bacteria can overpopulate your intestine or colon and can cause colitis.

Bacterial Reproduction (Pg 141 – 142)• Almost all bacteria reproduce by binary fission.

• In binary fission:– Cell elongates and DNA replicates.

– The two DNA strands are attached to the cell membrane and separated by new membrane growth.

– The new membrane pinches inward.

– The cell membrane thickens.

– The cell wall divides.

Generation Time • Generation Time - Is the interval of time

between successive binary fission of a cell.

• Escherichia coli generation time 20 minutes.

• Staphylococcus aureus 30 minutes.

• Mycobacterium tuberculosis 15 hours.

• Treponema pallidum (Syphilis) 33 hours.

How Many E. coli Would There Be After 2 Days?• E. coli generation time is every 20 minutes• 3 generations per hour• 48 hour period• N(t) =N(0)2n N(48) = (1) x 2144

• N(48) = 2144 • 2144 = 2.2 x1043

• 109 = 1,000,000,000• Each bacteria is 10-12 gms• The total bacterial mass from 2.2 x1043 = 1031

gms.• 454 gms = 1 pound• The mass would be

24,000,000,000,000,000,000,000,000 tons• or 400 x the mass of the earth

Bacterial Growth (Pg 143 – 149)

• Four distinctive phasesLag phaseLogarithmic phaseStationary PhaseDecline Phase

Lag Phase

• Bacteria adapts to its new environment.

• There is no cell division

• Bacteria:– Grow in size

– Store nutrients

– Synthesize enzymes

Logarithmic Phase (Log)

• Bacteria enter an active phase of growth.

• Exponential growth occurs by binary fission.

• In infection disease symptoms develop in animals or humans when the bacteria are in log phase.

– Toxin production causes tissue damage.

– Fever, coughing.

• However, bacteria are most susceptible to antibiotics in this phase because they are producing new cell walls.

Stationary Phase

• Point where death rate equals reproduction.

• Immune system is killing large numbers.

• Nutrients are running low.

• Waste products accumulate.

• Oxygen may be running out.

Death Phase

• Dying cells exceeds reproduction.

• Some cells, Bacillus and Clostridium, may have produced endospores and will enter dormancy.

• Others will end here.

• Bacterial Growth

Physical Requirements for Bacterial Growth• Temperature

– Each bacteria has an optimal growing temperature.

– Also, a 30ºC range at which growth can occur.

– Reduced growth is the result of slower reaction times for enzymes.

– Three Groups• Psychrophiles• Thermophiles• Mesophiles

Temperature

• Psychrophiles– Psychro = “Cold” Philes = “Loving”– Optimum growth rate 15ºC– Can grow at 0ºC - 20ºC– Unsaturated fats in phospholipids so they stay

fluid at lower temperatures.

• Thermophiles– Optimum temperature 60ºC– Growth at 40ºC - 70ºC– Compost heaps, hot tubs, hot springs– Highly saturated fats to stabilize structures

and heat stable enzymes.

Temperature

• Mesophiles– Middle temperature range 20ºC - 42ºC– Includes the pathogens– Get slow growth with some at low

temperatures 5ºC– Not true psychrophiles so referred to as

psychrotrophic– Psychrotrophic – grow at 0ºC but prefer to

grow in the range of 20ºC - 42ºC• Campylobacter jejuni can grow at low

temperatures

Oxygen• Aerobes – need a good supply of oxygen.

• Microaerophiles – live in areas of low oxygen.

• Anaerobes – do not and can not use oxygen.– Aerotolerant – can grow with oxygen present.– Obligate anaerobes are killed by any oxygen.– H2S not H2O – Clostridium species botulism gangrene.

• Facultative - grow in the presence or reduced oxygen.– Facultative aerobe prefers anaerobic conditions.– Facultative anaerobe prefers aerobic conditions.

• Capnophilic – low oxygen but high CO2

– Streptococcus are capnophilic

pH• In most bacterial cytoplasm is neutral pH 7.0.

• Blood and tissue of humans is 7.2 to 7.4 which is ideal for bacteria.

• Minimum to maximum pH range is between 3 pH units.

• Some can survive 2.0 pH or lower (acidic).

• Vibrio cholerae pH 2.0 to pH 9.5

Acidophiles

• Acidophiles are bacteria that can tolerate acidic conditions.

• Valuable to the food industry Lactobacillus and Streptococcus coverts milk to butter milk and cream to sour cream.

• Extreme acidophiles are found in the archaea bacteria.

• Most bacteria however do not survive in low pH

Measuring Bacterial Growth (Pg 155 to 156)

• Spectrophotometer measures turbidity (cloudiness).

– A spectrophotometer measures amount of light scatter (optical density, OD).

• Direct microscope count place culture on a specially designed slide (Petroff-Hausser counting chamber).

• Most probable number count (MPN) estimate number of cells by the amount of gas they produce through fermentation (breakdown of sugar).

• Total Plate Count bacterial culture is diluted and the different dilutions are plated and counted for colonies. – Colony forming units (CFU) we will do this next week in

hand scrub experiment

Summary of Key Concepts

• The Shape and Arrangements of Bacteria

– Bacilli a cylindrical shape

– Cocci form a variety of arrangements

– Spirals and other shapes also exist

The Structure of Bacteria• Bacterial flagella provide motility.

• Pili are structures used for attachment.

• The capsule (glycocalyx) is a sticky layer coating many bacteria.

• The cell wall provides shape and protection.

• The cell membrane is a permeability barrier.

• The cytoplasm is the center of biochemical activity.

• Endospores are designed for dormancy

• Bacterial reproduction– Bacteria reproduction by binary fission

• Bacterial growth– A bacterial growth curve illustrates the

dynamics of growth– Temperature is one of the most important

factors governing growth– Oxygen can support or hinder growth– Most bacteria prefer to grow at a neutral pH

• Culture media and growth measurements– Population growth can be measured in

several ways