b io 120 lecture 3 2012 2013

MICROBIAL CELL

BIOLOGY

Biology 120 Lecture 3

Reference: Chapter 3 and 4 TORTORA

Tuesday, July 3, 2012

GETTING TO KNOW YOUR BACTERIA

• Unit of measure (micrometers or nanometers)

• Microscopy = most essential tool in microbial cell biology


ESSENTIALS IN MICROSCOPY

• Simple versus Compound

• number of lenses

• Brightfield versus Darkfield

• background

• Light versus Electron Microscopy

• light/ beam of electrons

• SEM versus TEM

• 3D surface/ 2D internal structuresTuesday, July 3, 2012

BRIGHT & DARK


SEM & TEM


WHY OIO?


PREPARATION OF SPECIMENS FOR MICROSCOPY

WET MOUNT



FIXED SMEAR = STAININGTuesday, July 3, 2012


• FIXATION

• Process by which the internal and external structures of cells and microorganisms are preserved and fixed in position

• Inactivates enzymes that might disrupt cell morphology

• Toughens cell structures to prevent changes during staining and observation

• Usually microbes are killed when fixedTuesday, July 3, 2012

TYPES OF FIXATIONS• preserves overall

morphology but not structures within cells

• Penetrates cells and react with cellular components

• smear ready for staining!!!


STAINING

•STAINS

•salt of + or - ions, one of which is colored (chromophore)

•+ basic dye (CV, MB, MG, Sf)

•- acidic dye (Ng)

•Types of Staining: SIMPLE, DIFFERENTIAL, SPECIAL


POSITIVE AND NEGATIVE STAINING

CAN YOU SEE THE

DIFFERENCE?Tuesday, July 3, 2012

STAINING• SIMPLE

• e.g. MB, CF, CV, Sf

• highlight shapes and arrangements

• DIFFERENTIAL

• e.g. Gram stain and Acid fast stain

• differentiate and distinguish one kind of bacteria from another

• SPECIAL

• e.g. negative, endospore, flagella staining

• staining special structures


SIMPLE STAINING


GRAM STAINING


ACID FAST STAINING


NEGATIVE STAINING


ENDOSPORE STAINING


FLAGELLA STAINING


QUESTIONS?Tuesday, July 3, 2012

RECALL...• PROKARYOTIC ORGANISMS

• small, mostly unicellular

• bacteria and archaea

• DIFFERENCES & VARIATION

• bacteria vs archeae

• prokaryotes vs eukaryotes (microbes)

• FACTORS: morphology, chemical composition, nutritional requirements, biochemical activities, source of energy


THE PROKARYOTES


SIZE, SHAPE & ARRANGEMENT

OF BACTERIAL CELLSTuesday, July 3, 2012

WHAT ARE THE 3 BASIC SHAPES OF BACTERIA?

•Measure: 0.2-2.0µm (diameter) x 2-8µm (length)

•Basic shapes: coccus, bacillus, spiral


COCCI• usually round, can be oval, elongated or

flattened on one side

• e.g. diplococci (remain in pairs)

• e.g. streptococci (remain in chains)

• e.g. tetrads (remain attached in cube-like group of 4)

• e.g. sarcinae (remain attached in cube-like group of 8)

• e.g. staphylococci (divide in multiple planes, grape-like)


BACCILI

• divide only across their short axis; mostly single rods

• e.g. diplobacilli (remain in pairs after division)

• e.g streptobacilli (occurs in chains)

• e.g. coccobacilli (cocci-like)


SPIRALS & CURVED

• have on or more twists

• never staright

• e.g. vibrios (curved rods(

• e.g. spirilla (helical, cork-screw, rigid)

• e.g spirochetes (helical but flexible)


flagella - rigid

axial filament - flexibleTuesday, July 3, 2012

THE OTHERS

Stella sp. Haloarcula sp.


SHAPE = heredity* Monomorphic, maintain a single shape

** Pleomorphic more than one shape


ORGANIZATION IN A TYPICAL PROKARYOTE STRUCTURE:

1. Structures external to the cell wall

2. the cell wall

3. structures internal to the cell wall


STRUCTURES EXTERNAL TO THE

CELL WALLTuesday, July 3, 2012

GLYCOCALYX or SUGAR COAT• secreted on prokaryotic surface

• viscous, sticky, gelatinous polymer

• composed of polysaccharide, polypeptide or both

• made inside the cell and secreted outside

• CAPSULE: organized and firmly attached to the cell wall

• SLIMY LAYER: unorganized and loosely attached to the cell wall


FUNCTIONS:

• contributory to virulence (degree of pathogenicity)

• protect pathogen from phagocytosis

• attachment to various surfaces for survival

• prevent cell from dehydration

• viscosity = inhibits movement of nutrients out of the cell

• EPS (extracellular polysaccharide) = capsules made up of sugars


EXAMPLES

•Bacillus anthracis (anthrax)

•Streptococcus pneumoniae (bacterial pneumonia)

•Streptococcus mutans (dental caries)


OBSERVATION OF GLYCOCALYX

•Bacillus anthracis (anthrax)

•Streptococcus pneumoniae (bacterial pneumonia)

•Streptococcus mutans (dental caries)


FLAGELLA• long filamentous appendages that

propel bacteria

•3 basic parts:

•filament = long outermost region (flagellin)

•hook = where filament is attached (various protein)

•basal body = anchors flagellum to cell wall and plasma membrane


FLAGELLA

•atrichous = lacks flagellum

•monotrichous = single polar

•amphitrichous = tufts at both ends

• lophotrichous =two or more on one or both ends

•peritrichous = distributed over the entire cell


FLAGELLA


DO NUMBERS MATTER?


DIFFERENCES IN BASAL BODY (Gram + vs Gram -)


MOTILITY

•ability of an organism to MOVE by itself

•RUN-TUMBLE-RUN routine

•TAXIS: move away or towards a stimuls (chemotaxis, phototaxis)


FLAGELLA & MOTILITY


FLAGELLAR PROTEINS

•H antigen: useful for distinguishing among serovars of bacteria

•e.g. E. coli O157:H7

•NOTE: there are at least 50 fifferent H antigens for E. coli


AXIAL FILAMENTS•locomotory structure for

spirochetes

•also called “endoflagella”

•bundles of fibrils that arise at the ends of the cell beneath an outer sheath

•spirals around the cell


AXIAL FILAMENTS


FIMBRIAE & PILI

•hair-like appendages

•shorter, straighter and thinner than flagella

•used for attachment (F) and transfer of DNA/conjugation (P)

•essential for colonization in Neisseria (F)

•NOT for motility!


FIMBRIAE & PILI


THE CELL WALL


CELL WALL• complex, semi-rigid structure

• gives the shape of the cell

• surrounds the plasma membrane and protects interior from adverse changes in the outside environment

• prevents rupture of bacterial cells

• contributes to ability of some species to cause disease

• site of action of some antibiotics

• ALMOST ALL prokaryotes have cell walls


CELL WALL: Composition & Characteristics

• peptidoglycan or murein

• N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)

• Linked by polypeptides


CELL WALL: Composition & Characteristics


GRAM + CELL WALL


GRAM + CELL WALLNote: lipoteichoic acid

provide antigenic variation


GRAM - CELL WALL


GRAM - CELL WALLNote: outer membrane polysaccharides provide antigenic variation; Lipid

A is an endotoxin


GRAM + VS GRAM -


ATYPICAL CELL WALL

• e.g. Mycoplasma

• no walls or have little wall materials

• plasma membrane have sterols (help protect from lysis vice the CW)


ATYPICAL CELL WALL• e.g. Archaea

(Halobacterium sp)

• may lack walls or have unusual walls composed of polysaccharides and proteins not peptidoglycan (pseudomurein)

• PSEUDOMUREIN = lacks the D-amino acids found in bacterial CWs

• NOTE: Gram stain not applicable


ATYPICAL CELL WALL

• e.g. Mycobacterium and Nocardia

• high concentrations of mycolic acids in CWs (60%)

• prevents the uptakes of dyes

• Note: Gram stain will work only if mycolic acids removed


DAMAGING CELL WALL

• antimicrobial drugs (e.g. penicillin) = halts CW synthesis

• lysozyme = targets PG backbone


STRUCTURE INTERNAL TO THE

CELL WALLTuesday, July 3, 2012

PLASMA MEMBRANE• primarily phospholipids

• lacks sterols thus LESS rigid


PLASMA MEMBRANE


IMPORTANT STRUCTURES•GLYCOPROTEINS & GLYCOLIPIDS

•help protect and lubricate the cell

• involved in cell-to-cell interactions (e.g. pathogen binding in inlfuenza)


CM FUNCTIONS

•selective barrier

•breakdown of nutrients and production of energy

•What happens when CM destroyed?

•cell leakage


PHOTOSYNTHETIC STRUCTURES IN THE CM

purple non-sulfur bacteria

purple sulfur bacteria

green sulfur bacteria

PHOTOSYNTHETIC PIGMENTS IN MEMBRANE FOLDINGS:

1. Chromatophores2. Chlorosomes3. Photosynthetic lamellae


BACTERIA VS ARCHAEA CM

•EUBACTERIA

•Ester linkage

•Weaker linkage

•ARCHAEBACTERIA

•Ether linkage

•Stronger linkage


READING ASSIGNMENT: MEMBRANE TRANSPORT SYSTEMS


THE NUCLEAR AREA

• “NUCLEOID”

• contains the bacterial chromosome

• not surrounded by a nuclear envelope (membrane)

• do not include histones


THE NUCLEAR AREA• PLASMIDS

• extrachromosomal genetic element

• replicate independently

• Gene: antibiotic resistance, tolerance to toxic metals, toxin production and synthesis of enzymes

• can be transferred from one bacterium to another via conjugation


BACTERIAL RIBOSOMES

• ALL PROKARYOTES & EUKARYOTES HAVE RIBOSOMES!!!

• site of protein synthesis

• composed of two units:

• protein sub-unit

• ribosomal RNA subunit

• NOTE: differ from EUK ribosomes in the number of proteins and rRNA molecules they contain and they are less dense


BACTERIAL RIBOSOMES• PROK = 70S ribosomes while EUK = 80S

ribosomes

• The 70S = 30S (1 rRNA molecule) + 50S (2 rRNA molecules)

16S ribosomal DNA = prokaryotes

18S ribosomal DNA = eukaryotes


BACTERIAL RIBOSOMES• Antimicrobials:

• streptomycin and gentamicin = attach to 30S and interfere with protein synthesis

• erythromycin and chloramphenicol = attach to 50S and interfere with protein synthesis

• THUS only prokaryotes are affected by these antimicrobials


INCLUSIONS• inclusions = reserve deposits used when

supply are deficient

• METACHROMATIC GRANULES

• POLYSACCHARIDE GRANULES

• LIPID INCLUSIONS

• SULFUR GRANULES

• CARBOXYSOMES

• GAS VACUOLES

• MAGNETOSOMESTuesday, July 3, 2012

METACHROMATIC GRANULES

• “volutin”

• large inclusions

• stain red with certain blue dyes (e.g. MB)

• inorganic phosphate/polyphosphate reserves

• used for ATP synthesis

• BACTERIA, ALGAE, FUNGI & PROTOZOA

• Corynebacterium diphtheriae (diagnostic)Tuesday, July 3, 2012

METACHROMATIC GRANULES

“chinese characters”

diagnostic for C. diptheriae


POLYSACCHARIDE GRANULES

• consist of glycogen and starch

• demonstrated when iodine is applied to cells

• appear reddish brown (Glycogen)

• appear blue (Starch)


LIPID INCLUSIONS

• Mycobacterium, Bacillus, Azotobacter, Spirillum etc

• e.g. poly-B-hydroxybutyric acid (PHBs)

• revealed using Sudan dyes (fat soluble dye)


SULFUR GRANULES

• Thiobacillus spp, Beggiatoa

• they derive energy by oxidizing sulfur and sulfur-containing compounds

• deposit sulfur granules as energy reserves

Beggiatoa sp.


CARBOXYSOMES

• contain the enzyme 1,5-diphosphate carboxylase (for carbon dioxide fixation)

• photosynthetic bacteria, Nitrifying bacteria, Cyanobacteria, Thiobacillus


GAS VACUOLES

• hollow cavities in aquatic prokaryotes

• cyanobacteria, anoxygenic photosynthetic bacteria and halobacteria

• maintain buoyancy


MAGNETOSOMES• inclusion of iron oxide

• Magnetospirillum magnetotacticum

• used to move downward until they reacha suitable attachment site (act like magnets)

• can decompose hydrogen peroxide (to protect cells from its accumulation)


ENDOSPORES

• Clostridium, Bacillus (Bacteria)

• Thermoactinomyces vulgaris (Archaea)

• specialized resting cells

• resistant to adverse conditions (extreme heat, lack of water, exposure to toxic chemicals and radiation)

• Dipicolinic acid (DPA) with calcium ions directly involved in spore heat resistance


SPORULATION/SPOROGENESIS

SPORULATION: SPORE FORMATION

GERMINATION: SPORE TO VEGETATIVE CELL


ENDOSPORES


THE EUKARYOTES


EUKARYOTIC FLAGELLA & CILIA


For cellular locomotion

• Flagella: projections that are few and long in relation to the size of the cell (e.g. Euglena)

• Cilia: projections that are numerous and short in relation to the size of the cell (e.g. Tetrahymena)

• Difference between prokaryotic flagella:

• PROK = rotates

• EUK = moves in a wave-like manner


For cellular locomotion


HOW THEY PROPEL THE CELL


THE 9 + 2 ARRAY


EUKARYOTIC CELL WALL &

GLYCOCALYXTuesday, July 3, 2012

CELL WALLS

• EUK have simpler cell walls

• Algae: cellulose

• most Fungi: chitin

• Yeasts: glucan and mannan

• Protozoa: DO NOT HAVE a typical cell wall = pellicle (flexible outer protein covering)


GLYCOCALYX

• strengthens the cell surface

• helps attach cells together

• involved in cell to cell recognition


EUKARYOTIC PLASMA MEMBRANE


PLASMA MEMBRANE

• similar in function and basic structure with prokaryotes

• differences are the proteins found in the membranes

• also contain carbohydrates which serves as attachment sites for bacteria and as receptor sites for cell-to-cell recognition

• contains sterols (resist lysis due to osmotic pressure)


PLASMA MEMBRANE• NOTE: group

translocation do not occur in eukaryotic membranes

• instead ENDOCYTOSIS (e.g. pinocytosis and phagocytosis)


CYTOPLASM

• substance inside the plasma membrane and outside the nucleus

• cytosol = fluid portion of the cytoplasm


CYTOPLASM• Major difference:

• EUK have complex internal structures (microfilaments, intermediate filaments, microtubules) which forms the cytoskeleton (provides support for cytoplasmic streaming)

• many enzymes fund in cytoplasmic fluid of PROK are sequestered in the organelles of EUK


EUKARYOTIC RIBOSOMES & ORGANELLES


RIBOSOMES• same function as in PROK

• larger and denser than PROK (80S = 60S with 3 molecules of rRNA; and 40S with 1 molecule of rRNA)


RIBOSOMES• free ribosomes: unattached, protein

synthesis used inside the cell

• membrane-bound ribosomes: attached to nuclear membrane and ER, protein synthesis for insertion in the plasma membrane or for export from the cell

• polyribosome: located within mitochondria, synthesis of mitochondrial proteins (10-20 ribosomes joined together in a string-like arrangement)


ORGANELLES

• organelle: structure with specific shapes and specialized functions; absent in prokaryotes

• Nucleus, ER, golgi complex, lysosomes, vacuoles, mitochondria, chloroplasts, peroxisomes and centrosomes


ORGANELLES

•Nucleus = houses the chromosome

•ER = transport and storage

•Golgi complex = membrane foration and protein secretion

•Lysosomes = store digestive enzymes

•Vacuoles = storage and rigidity


ORGANELLES• Vacuoles = storage and rigidity

• Mitochondria = site of ATP production

• Chloroplasts = contain chlorophyll and enzymes for photosynthesis

• Peroxisomes = oxidation of organic compiunds (e.g. catalase) destroying hydrogen peroxide)

• Centrosomes = contains centrioles for mitotic spindle formation


ORGANELLES


THE EVOLUTION OF EUKARYOTES


HOW THEY CAME ABOUT...

• 3.5-4B years ago = simple organisms (similar to prokaryotes)

• 2.5B years ago = eukaryotes from prokaryotes

• Lyn Margulis: The Endosymbiotic Theory

• larger bacterial cells lost their CW and engulfed smaller bacterial cells

• endosymbiosis = lives within another


ENDOSYMBIOTIC THEORY

• ancestral EUK developed a rudimentary nucleus when the plasma membrane folded around the chromosome (NUCLEOPLASM)

• Nucleoplasm ingested aerobic bacteria and lived inside it

• evolved into a symbiotic relationship (host supply nutrients, while bacteria produce the energy from supplied nutrients


ENDOSYMBIOTIC THEORY

• CHLOROPLASTS = descendants of photosynthetic prokaryotes ingested by the nucleoplasm

• FLAGELLA & CILIA = motile spiral bacteria/spirochetes


NEXT MEETING: JOURNAL REPORTING


b io 120 lecture 3 2012 2013

Technology

cell structures

cell morphology

cell wall2

cell wall3

simple stainingtuesday

negative stainingtuesday

gram stainingtuesday

endospore stainingtuesday