introduction structure

MICROBIOLOGY

FALL 2011

Office Hrs. MW 11-12; STC 239Tue 10-1; NEWDL 312 Independence Rd.

Laboratory M/W 12-3Lecture MW 3-4:15

Syllabus

Dasek et al. 2000

Human Pathogens

1415 known human pathogens 61% are zoonotic

Wild Animals Involved in Wild Animals Involved in MPXV Transmission (USA, 2003)MPXV Transmission (USA, 2003)

Gambian Giant Rat(Crycetomys sp.)

Prairie Dog(Cynomis sp.)

Ghana

TX

WI

IL*

IA

15/4

09/04

Entry and Distribution Route of Entry and Distribution Route of Crycetomys spCrycetomys sp. and . and Cynomys sp.Cynomys sp. Involved in MPXV Outbreak (USA, 2003) Involved in MPXV Outbreak (USA, 2003)

Source: MMWR 52 (23), CDC, 2003.

15/4

Index case in Marshfield, 26 May 2003:

Disseminated lesions.

Distribution of MPXV Cases in the USA

WI

IL IN OH

KSMO

Number of cases = 87WI = 38IN = 24IL = 19

OH = 4KS = 1MO = 1

Source: MMWR 52 (23), CDC, 2003.

Microbiology

Has been defined as the study of organisms and agents too small to be seen clearly by the naked eye – that is, the study of microorganisms

Scope and Relevance Microorganisms are exceptionally diverse, are found almost everywhere, and affect human society in countless ways. Modern microbiology is a large discipline with many different specialties: medicine, agricultural & food sciences, ecology, genetics, and biochemistry.

Microbiology

V iro log y B ac te rio log y H e lm in th o log y P ro tozo log y P h yco log y M yco log y

M ic rob io log y

Microbiology

P ath o log y Im m u n o log y G en etics M olecu la r B io log y

M ic rob io log y

Microbiology

Spontaneous Generation Molecular Biology/Genetics Fermentation Infectious Disease

Infectious Disease - History

1798 Jenner, smallpox vaccine 1835-1844 Bassi, silkworm disease

1847-1850 Semmelweis 1849 Snow, cholera 1861 Pasteur disproves spontaneous generation

1867 Lister antiseptic surgery

Infectious Disease - History 1876-1877 Koch anthrax is caused by Bacillus anthracis

1881 Pasteur develops anthrax vaccine

1884 Koch’s postulates publishedAutoclave developedGram stain developed 1885 Pasteur develops rabies vaccine


1887 Petri dish developed 1890 Von Behring prepares antitoxins for diphtheria and tetanus

1899 Ross shows that mosquitoes carry malaria

1910 Ehrlich – magic bullet 1923 – Bergey’s Manual 1st edition


1929 Fleming discovers penicillin

1935 Domagk discovers sulfa drugs

1975 Lyme Disease 1983 HIV 1986 hepatitis B vaccine – genetically engineered

DISEASE

Occurrence of Disease

Signs & Symptoms of Disease

Fever Fatigue White Blood Cell Count Pain Muscle Aches Blood Pressure

Molecular Biology

1941 Beadle and Tatum, one gene-one-enzyme hypothesis

1944 Avery shows that DNA carries information during transformation

1952 Hershey and Chase viral infection of bacteria

1953 Watson & Crick

Molecular Biology - History

1961 Jacob & Monod propose the operon model of gene regulation

1961-1966 Nirenberg et al. elucidate the genetic code

1970 Arber & Smith – restriction endonucleases

2000 Human genome sequenced

Fermentation - History

1857 Pasteur shows that lactic fermentation is due to a microorganism

1897 Buchner prepares yeast extract that ferments

Spontaneous Generation

1799 Spallanzani attacks spontaneous generation

1861Pasteur disproves spontaneous generation

Kingdoms

Animal Plant Fungi Protista Prokaryotic

Classification

Kingdom Phylum Class Order Family Genus species

Infectious Agents / Microrganisms

Bacteria Fungi Prions Protozoans Helminths

Toxins – Exo and Endotoxins

Prokaryotes Greek – before a nucleus

Prokaryotes / Eukaryotes

There are a number of differences between bacterial cells and plants, animals, fungi and protozoans.


Size of cell

1-10um diameter

10-100um diameter

Nucleus No nuclear membrane

True nucleus

Membrane bound organelles

absent present

Flagella 2 protein building blocks

complex


Glycocalyx Slime layer Absent

Cell Wall Usually presentComplex

When present, simple

Plasma membrane

No CHO and lack sterols

Sterols and CHO present

Cytoplasm No cytoskeleton

cytoskeleton


Ribosomes Small size 70s

Large 80s; small 70s

Chromosome Circular, lacks histones

Linear, with histones

Cell division

Binary fission

mitosis

Sexual reproduction

No meiosis meiosis

Bacterial Classification

Cellular Characteristics Morphology – cell shape, cell size, arrangement of cells, arrangement of flagella, capsule, endospores

Cellular Characteristics

Staining Reactions – Gram stain, acid-fast stain


Growth and nutritional characteristics – appearance in liquid culture


Growth and nutritional characteristics–colonial morphology


Growth and nutritional characteristics pigmentation


Growth and nutritional characteristics energy sources, C, N sources,


Growth and nutritional characteristics fermentation products


Growth and nutritional characteristics modes of metabolism


Biochemical Characteristics – cell wall constituents, pigment biochemicals, storage inclusions, antigens, RNA molecules


Physiological and Ecological Characteristics – temperature range and optimum


Physiological and Ecological Characteristics oxygen relationships


Physiological and Ecological Characteristics – pH tolerance range


Physiological and Ecological Characteristics –salt requirement and tolerance


Genetic Characteristics- DNA G + C

DNA hybridization

Fimbriae & Pili

G- bacteria have short, fine, hairlike appendages that are thinner than flagella and not involved in motility

Fimbriae & Pili

slender tubes composed of helically arranged protein subunits and are about 3 to 10 nm in diameter and up to several um long

FIMBRIA (s) FIMBRIAE (pl)

Composition varies, contain protein

Tendency to stick to each other and surfaces Bacterial attachment in aqueous environments

Role in colonization infection

PILUS (s) PILI (pl)

Hollow, non-helical (9-10nm dia)

Filamentous appendages

Thinner than flagella, more numerous

Example F-pilus (SEX PILUS)

entry of genetic material during conjugation

GRAM -VE BACTERIA ONLY

E.M Pili on E. coli – N. gonorrhea

Pili & Fimbriae Some types of fimbriae attach bacteria to solid surfaces such as rocks in streams and host tissues

Pili – about 1 to 10 per cell, differ from fimbriae: are larger (9 to 10 nm in diameter), they are genetically determined by sex factors or conjugative plasmids and are required for bacterial mating

Pili & Fimbriae

Some bacterial viruses attach specifically to receptors on sex pili at the start of their reproductive cycle

Flagella & Motility

Most motile bacteria move by use of flagella, threadlike, locomotor appendages extending outward from the plasma membrane and cell wall.

Slender, rigid structures, about 20 nm across and up to 15 to 20 um long

Flagella - Arrangements

Monotrichous Amphitrichous Lophotrichous Peritrichous

Flagellar Ultrastructure

Filament Hook Basal body

ARRANGEMENT OF FLAGELLA POLAR - at one or both ends

MONTRICHOUS - single e.g., Vibrio sp.

LOPHOTRICHOUS - small tufts at same site

e.g., Pseudomonas sp.

AMPHITRICHOUS - at both poles, e.g., Spirillum sp.

LATERAL

PERITRICHOUS - surrounding entire cell, e.g., Proteus sp.

MOTILITY CHEMOTAXIS: movement towards/away from chemicals

MAGNOTAXIS: orientation of movement in magnetic field

Aquaspirillium magnetotacticum - Magnetosomes

(Fe3O4 crystalline magnetic iron oxide)

PHOTOTAXIS: Differences in light intensity THERMOTAXIS: heat

Rotation of flagellar motor: reversible

Clockwise (CW) or Counterclockwise (CCW)

Smooth swimming/running motion

Motor rotates CCW direction

Flagella sweep around cell (in common axis)

Tumbling motion

Motor reverse (CW direction)

Flagella disperse

Flagellar Synthesis Complex process – involving at least 20 – 30 genes

Flagellin subunits are transported through the filament’s hollow internal core.

When they reach the tip, the subunits spontaneously aggregate so that the filament grows at its tip rather than at the base

Self - assembly

The Mechanism of Flagellar Movement

The filament is in the shape of a rigid helix, and the bacterium moves when this helix rotates


Act like propellers on a boat


E. coli rotates 270 r.p.s., Vibrio alginolyticus averages 1,100 r.p.s.

Distance and speed:

20-90m/sec

Equivalent to:

6ft human running 5 body lengths/second

AXIAL FILAMENTS

Modified flagellum

Long thin microfibril, inserted into a hook, entire structure enclosed in periplasmic space

ENDOFLAGELLUM

CELL SURFACE

3 Basic Layers - GLYCOCALYX CELL WALL CELL MEMBRANE

collectively termed CELL ENVELOPE

Glycocalyx Is a network of polysaccharides extending from the surface of bacteria and other cells

Aids in bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in plant and animal hosts

GLYCOCALYX

External mucilaginous layer Surrounds cell Shows organisation

SLIME LAYER - abundant, easily washed off

(poorly organised)

CAPSULE - abundant, not easily washed off

Capsule Well organized and not easily washed off

Composed of polysaccharides, but may be constructed of other materials

i.e. Bacillus anthracis has a capsule of poly-D-glutamic acid

Visible with the light microscope Resist phagocytosis

Capsules

Contain a great deal of water – protect cell from desiccation

Exclude viruses Exclude most hydrophobic toxic substances

FUNCTIONS Provide protection (drying)

Block attachment of bacteriophages

Antipathogenic (inhibit engulfment of pathogenic bacteria by WBC’s) Contributes to VIRULENCE or INFECTIVE ABILITY

Promote attachment to surfaces

Streptococcus mutans: adheres to teeth (GLUCAN), DENTAL CARIES

Complement Activation Some capsules prevent formation of C3 convertase on the bacterial surface

Host response – Antibody Subvert this type of protective host response by having capsules that resemble host polysaccharides.

CELL WALL

Important in bacterial characteristics

Determines shape

Provides support/rigidity

STRUCTURE Composed of PEPTIDOGLYCAN (MUREIN)

Insoluble, porous

Cross-linked polymer (glycan), provides strength and rigidity

N-acetyl Muramic Acid (NAM)

N-acetyl Glucosamine (NAG)

1-4 glycosodic bonds

Differences in structure - basic principles of

GRAM STAIN REACTION

Christian Gram 1884Differential stain: ability of eubacterial cells to retain dye

(crystal violet) after discolouration with 95% ethanol

Cells retain stain: Gram +ve (thick cell walls) PURPLE

Cells lose stain: Gram -ve (thin cell walls) RED

Peptidoglycan Structure

Peptidoglycan or murein is an enormous polymer composed of many identical subunits.

Peptidoglycan Structure Most G- cell wall peptidoglycan lacks the peptide bridge.

Gram Negative Cell Walls More complex than the G+ cell walls. Peptidoglycan 5-10% of the wall weight.

Braun’s lipoprotein – a small lipoprotein covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end.

Lipopolysaccharides (LPSs)

Lipopolysaccharide Structure

Known as ENDOTOXIN

Complex molecule: Inner most LIPID (Lipid A), achors LPS to outer membrane

Polysaccharide portion (external to Lipid A) known as O-antigen

O-polysaccharide long repeating sequence of sugars

LPSs Contain both lipid and carbohydrate

Consist of three parts: lipid, the core polysaccharide and the O side chain

LPSs The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or pyrophosphate attached.

LPSs Lipid A is buried in the outer membrane and the remainder of the LPS projects from the surface.

LPS

The core polysaccharide is joined to lipid A. In Salmonella it is constructed of 10 sugars, many of them unusual in structure.

LPS

The O side chain or O antigen is a short polysaccharide chain extending outward from the core. It has several peculiar sugars and varies in composition between bacterial strains.

LPS G- bacteria can rapidly change the nature of their O side chains to avoid detection.

Contributes to the negative charge of the bacterial surface

LPS helps stabilize membrane structure

Lipid A is toxic – endotoxin Serves as a protective barrier

LPS – protective barrier Prevents or slows entry of bile salts, antibiotics, and other toxic substances.

Porin Proteins Cluster together and span the outer membrane to form a narrow channel through which molecules smaller than about 600 to 700 daltons can pass.

.

Porin Proteins Larger molecules such as vitamin B12 must be transported across the outer membrane by specific carriers. The outer membrane also prevents the loss of constituents like periplasmic enzymes.

COMPARISON OF GRM+VE /GRM-VE CELL WALLS

CHARACTER +VE -VE

No’ of major layers 1 2

Chemical Make-up PeptidoglycanTechoic acid

Lipotechoic acid

LipopolysaccharideLipoprotein

Peptidoglycan

Overal Thickness Thick (20-80nm) Thin (8-11nm)

Outer Membrane No Yes

Periplasmic Space In some In all

Porin Proteins No Yes

Permeability More Permeable Less Penetrable

Periplasmic Space

Contains enzymes involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell.

Periplasmic Space Gram – contains many proteins that participate in nutrient acquisition. Ex. hydrolytic enzymes attacking nucleic acids and phosphorylated molecules, and binding proteins involved in transport of materials into the cell.

Denitrifying and chemolithoautotrophic bacteria – electron transport proteins in periplasm

CELL/CYTOPLASMIC MEMBRANE

4-5nm thick

composed 1o phospholipids 30-40%

and protein 60-70%

Phospholipid bilayer: Polar heads

(outwards into aqueous phase - membrane surface)

Fatty acyl tails

(inwards - semi/liquid phase at interior)

FLUID MOSAIC MEMBRANEDavison & Danelli

ALSO CONTAINS:

PROTEINS

INTEGRAL - removed by destruction

i.e., Detergents

PERIPHERAL - loosely attached, easily removed

i.e., Osmotic shock

FUNCTION

Transport - control nutrients Oxidative phosphorylation (Respiration) Secretion - discharge of metabolic products Anchoring DNA (during cell division) Metabolism - enzyme sites

INTERNAL CONTENTS

Cell Material divided into

PROTOPLASM

Granular appearance

Site of biochemical activity

Water 70-80%

acts as solvent for nutrients, sugars, Aa’s & salts

CHROMATIN AREA

no distinct membrane enclosed nucleus

no mitotic apparatus BACTERIAL CHROMOSOME

Typically single circular strand of DNA (CHROMATIN BODY)

Exception Streptomyces & Borrelia sp (Linear)

Rhodobacter sphaeroides (2 separate chromosomes)

all genes are linked

Aggregated in one area (NUCLEOID)

Bacterial Chromosome

PLASMIDS

Additional to chromosome

1 or more, small circular macromolecules of DNA

Capable of self-replication

Types:

Fertility (F-plasmid): genes for mating in conjugation

Resistance (R-plasmids): antibiotics, metals

Virulence factor: enterotoxin, fimbriae, antibiotic production

Colicinogenic (col-plasmids): gene for protein (COLICINS) toxic to closely related bacteria (eliminates competitors)

Transformation (Ti-plasmids): plant microbiology (formation of crown gall tumors) Agrobacterium

Metabolic: utilization of camphor, toluene

OTHER FEATURES

RIBOSOMES

Located in Protoplasm

RNA/PROTEIN bodies

Composed of 2 sub units (70S)

Svedberg Units

Sites of Protein Synthesis

MESOSOMES:

Extensive invaginations (infoldings) of cyto membrane

Continuous with membrane

Function NOT KNOWN

Corynebacterium parvum

INCLUSIONS/VACUOLES

compensate for poor availability of nutrients

present in Protoplast

VOLUTIN GRANULES/METACHROMATIC (coloured)

composed of POLYPHOSPHATE

energy rich storage structures

VOLUTIN

i.e., POLY--HYDROXYBUTYRATE

serve as carbon and energy source

METACHROMATIC

i.e., Aquatic bacteria - colored crystals

(Blue or Red dyes)

e.g., Corynebacterium

Cyanobacterium: Microcystis (12,600x)

Gas vacuoles - blue Storage granules - red

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