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Sophomore Dental andOptometry Microbiology:

Bacterial Structure andPhysiology

Janet Yother, Ph.D.Department of Microbiology

jyother@uab.edu4-9531

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Relevant Chapters

• Murray - 2, 3, 4• Jawetz - 2, 3, 4, 5

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Domains (Kingdoms)Based on evolutionary relationships

• Eukaryote (Plants, Animals, Protists, Fungi)• Eubacteria (Eubacteria)• Archaebacteria (Archaebacteria)

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Bacterial Nomenclature• Kingdom Eubacteria• Division Gracilicutes• Class Scotobacteria• Subclass• Order Spirochaetales• Family Spirochaetaceae• Tribe• Genus Borrelia• Species Borrelia burgdorferi

– Subspecies

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Eukaryotes - nuclear membrane (true nucleus)

• Animals• Plants• Protists - simple eukaryotes (Algae, Fungi, Protozoa)

Prokaryotes - no nuclear membrane (primitive nucleus)

• Eubacteria - true bacteria. Includes most bacteria.

• Archaebacteria - primitive. Evolutionarily separated.methanogens - produce methanehalophiles - grow in high saltthermophiles - grow at high temp

Differences in cell walls (lack PG), membranes (ether- rather than ester-linked lipids), ribosome components and metabolism. Share somefeatures with eukaryotes (introns, histones, etc).

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Distinctive Features of Prokaryoticand Eukaryotic Cells

Lacks functions ofprokaryotic membrane

Respiration, secretion,macromolecular synthesis

Cytoplasmic Membrane

80S (60S + 40S)70S (50S + 30S)Ribosomes

Usually presentAbsent (except in Mycoplasma)Sterols

No peptidoglycan(cellulose, chitin in some)

Peptidoglycan (absent inMycoplasma)

Cell Wall

Mitochondria (andchloroplasts inphotosynthetic organisms)

NoneOrganelles in CytoplasmIn organellesOften present (plasmids, phage)Extrachromosomal DNA

Membrane-bound; anumber of individualchromosomes

No membrane; single, (usually)circular chromosome

Nucleus

EukaryotesProkaryotesCell component

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• Prokaryotes - lack nuclear membrane (unlike

eukaryotes)

• Single-celled

• Reproduction - simple division, i.e. binary fission

• Small, ~1 µm (mycoplasmas as small as 0.2 µm;

bacillus as large as 10 µm)

• Various shapes (cocci, rods, spiral) and

arrangements (chains, clusters)

• Most are free-living, a few (rickettsiae,

chlamydiae) are obligate intracellular parasites

BACTERIA

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Prokaryotic Cell Morphology

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BACTERIAL CELL

• 50% protein• 20% nucleic acids (10x more RNA than

DNA)• 10% polysaccharides• 10% lipids

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Bacterial Chromosomes

• Single, circular, double-stranded DNA(exception - borrelia = linear)

• Haploid (1 to 4 copies depending on growth rate)

• 600 to 4500 kb* in size. Smaller = moredependent on host/environment

• Up to 1 mm in length; supercoiled• Contained in nucleoid

* ~1 kb/gene

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Bacterial Nucleoids• Contain chromosomal DNA (60%; 2-3% dry wt of

cell); RNA (30%); Protein (10%)

• No nuclear membrane• No histones; ~6 chromosome-associated basic

proteins involved in determiningchromosomal structure

• Polyamines, e.g., spermidine and putrescine,neutralize negative charges on phosphates

• Haploid chromosome in cytoplasm– 1 to 4 nuclear bodies/cell, number depends on

growth rate (faster = more)• Can be membrane-associated (cell division)

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Extrachromosomal DNA

• Plasmids - Replicate in cytoplasm,independent of chromosome.– Usually circular (borrelia = linear)– Few to several hundred kb.– Conjugative (F, R), antibiotic resistance, metabolic,

virulence

• Bacteriophage - virus;– replicates in cytoplasm or integrates into

chromosome– can contribute to virulence

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Cytoplasmic Membrane• Lipid bilayer

– Permeability barrier– Active transport– Electron transport– Oxidative phosphorylation– Photosynthesis

• Affected by antibacterials– Detergents– Polymyxins (damage PE-

containing membranes)– Ionophores (disrupt

membrane potential)

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Cell Wall

• Shape• Barrier (osmotic

resistance)• Comprised of highly

crosslinked peptidoglycan• Affected by antibacterials

(e.g, β-lactam antibiotics,lysozyme)

• Basis for gram-stain

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Peptidoglycan

• Backbone of N-acetylglucosamine and N-acetyl muramic acid

• Cross-linked bypeptide bridges atMurNAc

http://employees.csbsju.edu/hjakubowski/classes/ch331/cho/peptidoglycan.gif

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Peptidoglycan

http://de.wikipedia.org/wiki/Peptidoglycan

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Peptidoglycans

[GlcNAc-MurNAc]n

L-ala

D-glu

L-lys (gly)n

D-ala

[GlcNAc-MurNAc]n

L-ala

D-glu

L-lys (gly)n

D-ala

Transpeptidases (TP) link.

Hydrolases (lysosyme, mutanolysin, e.g.) cleaveAmidases (autolysins, e.g.)cleave

PG structures varybetween/among Gm+and Gm-. This = Gm+.

β-lactams resemble TP substrates, block crosslinking of growing chain

Transglycosylases (TG) link

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β-lactams and PeptidoglycanCrosslinking

Transpeptidase

[GlcNAc-MurNAc]n

L-ala

D-glu

L-lys

D-ala

D-alanon-crosslinked peptidoglycan

CH3HC

CHCH3

CHN

ONH

HOOC

Terminal D-ala-D-ala

β-lactamring

CH2C ONHCHCH

(CH3)2HOOC

CN

O

HC C S

Benzylpenicillin(penicillin G)

R

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Gram Stain • Gram’s crystal violet (CV) • Potassium-iodide (KI) • Ethanol - decreases hydration of cell wall • Wash

⇒ CV-I complexes trapped in thick cell walls (cells remain purple = gram-positive)

• Safranin (red)⇒ thin cell walls don’t retain CV-I complexes,

counterstained with safranin(red = gram-negative)

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Exceptions to gram-positive /gram-negative staining

• Mycoplasmas - no cell wall.• Mycobacteria - lipid interferes with stain

– Detected with acid fast stain (carbol fuschinretained following decolorization withHCl/EtOH)

Both are related to gram-positives, based ongenetic analyses (rRNA sequence)

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Gram-positives

• Cytoplasmic Membrane• Cell wall• Lipoteichoic acid• Teichoic acid• Proteins

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Gram-positives• Cell Wall

– Thick peptidoglycan (10 to 100 nm)– Wall teichoic acids (WTA) - repeating units of phosphodiester-

linked glycerol or ribitol backbone + side chains (D-ala, glucose).Covalently linked to PG.

• Lipoteichoic acids (LTA) - membrane-anchored, structuremay differ from WTA

Staphylococcus aureus(Ribitol-P; 30-50 repeats)

CH2OH

H-C-O-R O

H-C-O-R P

H-C-O-R O-

CH2O

O-CH2

H-C-O-R

H-C-O-R

H-C-O-R

CH2O

R = GlcNAc or Ala

R-O-C-H P R-O-C-H P

CH2OH O O-CH2 O O

CH2O O- CH2O O

-esterlinkage

Phosphodiester(-) charge

R = Ala or glucose

Bacillus subtilus Marburg(1,3-linked Glycerol-P)

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Gram-positives• Cell Wall

– Thick peptidoglycan (10 to 100 nm)– Wall teichoic acids (WTA) - repeating units of phosphodiester-

linked glycerol or ribitol backbone + side chains (D-ala, glucose).Covalently linked to PG.

• Lipoteichoic acids (LTA) - membrane-anchored, structuremay differ from WTA

• LTA and WTA - ion binding, charge maintenance, membrane integrity, adherence, anchor proteins

• Cell walls - inflammation

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Gram-positives• May have polysaccharide capsule covalently linked to PG

Glc-AATGal-GalNAc-GalNAc-O-CH2

O H-C-OH

O-P=O O H-C-OH

(CH3)3-H-CH2-CH2-O O-P=O H-C-OH

(CH3)3-H-CH2-CH2-O C-O-P O-CH2

O-

+

+ =

O

phosphocholine

Oligosaccharide-Ribitol-P

(n=6-8)

O

O OH

peptideHNAc

O

CH2OHO

O

HNAc

C6

--GlcNAc----MurNAc----GlcNAc----MurNAc--

TEICHOIC ACID PEPTIDOGLYCAN

autolysin

mutanolysin

O

peptideHNAc

O

CH2

CAPSULE

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Gram-negatives

• Cytoplasmicmembrane

• Cell Wall• Outer membrane• Lipopolysaccharide• Proteins

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Gram-negatives

• Cell Wall– Thin peptidoglycan (1 layer; 2 nm)– No WTA or LTA

• Periplasmic space - digestive and protectiveenzymes; transport

• Outer membrane (OM) - blocks entry of largemolecules (>800 Da). Not typical lipid bilayer.– Attached to PG by lipoprotein– Lipopolysaccharide (LPS) - forms outer leaflet of OM– OM proteins - transport, porins

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Lipopolysaccharide (LPS)• Endotoxin - toxic shock; fever. leukopenia, hypotension,

acidosis, DIC, death

(OM)-Lipid A --- core polysaccharide --- O Agtoxic properties varies with species polysaccharide

varies with strain3 - 4 sugars/repeatUp to 25 repeatsserotyping

MM LM

HM HM

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Optional Features (Gram +/-)• Capsules - polysaccharide or protein

– Antiphagocytic (block C3b deposition or recognition),attachment

• Surface Proteins - anchored in CM, OM, CW– Antiphagocytic, attachment

• Flagella - protein. Rotates to propel cell.– Motility, chemotaxis, virulence (H-antigen)

capsules - colony

capsules - microscope

Flagella - EM

Flagella - peitrichous

Flagella - unipolar

Optional Features (Gram +/-)

• Pili - protein. Shorter, narrower than flagella.• Common - peritrichous; attachment• F (sex) - single; gene transfer (conjugation; gram -)

• Toxins - excreted; act on host cells; Clostridiumbotulinum; Vibrio cholerae

• Enzymes - hyaluronidase, proteases, DNases• Endospores - dehydrated cells; Clostridium, Bacillus

species (gram +)

F-pilus

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Growth Requirements

• Water - 70 to 80% of cell• Carbon and energy source (may be same)

– Most bacteria, all pathogens = chemoheterotrophs (useorganic molecules for carbon and energy sources)

– monosaccharides - glucose, galactose, fructose, ribose– disaccharides - sucrose (E. coli can't use), lactose (S.

typhimurium can't use)– organic acids - succinate, lactate, acetate– amino acids - glutamate, arginine– alcohols - glycerol, ribitol– fatty acids

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Growth Requirements - Nitrogen

• Inorganic source– Ammonia (NH4

+) → glutamate, glutamine– Nitrogen fixation N2 → NH4

+ → Glu, Gln– Nitrate (NO3

-) or nitrite (NO2-)

• Nitrate reduction NO3 → NO2 → NH4+

• Denitrification NO3 → → N2 (use NO3 as electronacceptor under anaerobic conditions, give off N2)

• Organic source– amino acids, e.g. (Glu, Gln, Pro)

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Growth Requirements - Oxygen

• Aerobe (strict) - requires O2– Cannot ferment (i.e., transfer electrons and protons

directly to organic acceptor); always transfers tooxygen (respires)

• Anaerobe (strict) - killed by O2– lack enzymes necessary to degrade toxic O2

metabolites; always ferment

superoxide radical

O2 2H2O2 2H2O + O2flavoproteins catalase

2O2 2O2- O2 + H2O2

Ferrous ion + 2H+

TOXIC

hydrogen peroxide

superoxide dismutase hydrogen peroxide

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Growth Requirements - Oxygen

• Aerobe (strict) - requires O2– Cannot ferment (i.e., transfer electrons and protons

directly to organic acceptor); always transfers tooxygen (respires)

• Anaerobe (strict) - killed by O2– lack superoxide dismutase, catalase; always ferment

• Facultative - grows + or - O2 (respire or ferment)• Aerotolerant anaerobe - grows + or - O2 (always

ferments)• Microaerophilic - grows best with low O2; can

grow without

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Growth Requirements

• Temperature– Thermophiles - >50oC– Psychrophiles - 4oC to 20oC– Mesophiles - 20oC to 40oC

• pH - mostly 6 to 8; can vary with environment• Other

– Sulfur, phosphorous, minerals (K, Mg, Ca, Fe), growthfactors (aa, vitamins)

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Nutrient Uptake

1. Hydrolysis of nonpenetrating nutrients byproteases, nucleases, lipases

2. Membrane transport - protein mediateda. facilitated diffusion b. active transport - group translocationc. active transport - substrate translocation

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Facilitated Diffusion

• Passive mediated transport• No energy required• Carrier protein equilibrates [substrate]

in/out of cell• Phosphorylation traps substrate in cell• Glycerol = example

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Active Transport - Grouptranslocation

• Requires energy (PEP, ATP)• Carrier protein concentrates substrates in

cell• Substrate altered and trapped in cell• Glucose = example

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Active Transport - SubstrateTranslocation

• Requires energy (proton gradient or ATP)• Carrier protein concentrates substrate in cell• Substrate unchanged. Transport system has

higher affinity for substrate outside cell.

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Bacterial Growth in Culture• Lag phase - actively

metabolizing; gearing up foractive growth

• Log phase - exponential growth• Stationary phase - slowed

metabolic activity and growth;limiting nutrients or toxicproducts

• Death phase - exponential lossof viability; natural or inducedby detergents, antibiotics, heat,radiation, chemicals

lag

exponential (log)

stationary

death

time, hr

log C

FU/m

l

log O

D

O

R

Growth rate dependent on bacterium, conditionsMaximum attainable cell density ~1010/ml (species-dependent)

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Bacterial Taxonomy

• Classification - arrangement into taxonomicgroups based on similarities andrelationships.

• Nomenclature - assignment of names byinternational rules. Yersinia pestis, Y. pestis

• Identification - determine group to whichnew isolate belongs

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Numerical Classification -enumerates similarities and differences

• Morphology– Microscopic - size, shape, motility, spores,

stains (gram, acid fast, capsule, flagella)– Colony - shape, size, pigmentation

• Biochemical, physiological traits - growthunder different conditions (sugars, C, pH,temp, aeration)

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Serological Classifications

• Reactivity of specific antibodies withhomologous antigens of different bacteria

• Usually surface antigens - capsules, flagella,LPS (O Ag), proteins, polysaccharide, pili

• Important in epidemiology

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Genetic relatedness

• Multilocus enzyme electrophoresis• Ability to exchange and recombine DNA• DNA restriction profile• DNA base composition - %GC

– Very different - unrelated– Very similar - may be related

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Multilocus Enzyme Electrophoresis1 2 ref

Starch gel; enzyme assays to detect proteins, shifts in mobility

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Genetic relatedness

• Multilocus enzyme electrophoresis• Ability to exchange and recombine DNA• DNA restriction profile• DNA base composition - %GC

– Very different - unrelated– Very similar - may be related

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RFLP-analysis

DNACut with restriction

enzyme

1 2 3 4

Agarose gel stained with ethidium bromide

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Genetic relatedness

• Multilocus enzyme electrophoresis• Ability to exchange and recombine DNA• DNA restriction profile• DNA base composition - %GC

– Very different - unrelated– Very similar - may be related

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Genetic relatedness

• DNA sequence - genes, whole genomes; true %identity

• DNA hybridization - total or specific sequences• DNA-RNA homology - hybridization between

DNA and rRNA (highly conserved, small part ofgenetic material)

• rRNA sequence - most useful– Determine sequence of DNA encoding rRNA

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DNA Hybridizationss DNA

Total or specific gene

+ 3H labeled DNA (ss) from unknowns

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DNA Hybridization - PCR

http://www.246.ne.jp/~takeru/chalk-less/lifesci/images/pcr.gif

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Genetic relatedness

• DNA sequence - genes, whole genomes; true %identity

• DNA hybridization - total or specific sequences• DNA-RNA homology - hybridization between

DNA and rRNA (highly conserved, small part ofgenetic material)

• rRNA sequence - most useful– Determine sequence of DNA encoding rRNA

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Sensitivity of rRNArRNA - associated with ribosome; critical for protein

synthesis(DNA ------------> mRNA -------------> protein)

• binds initiation site (Ribosome binding site, Shine-Delgarno sequence) in mRNA

• must have 2o structure (base pairs with self)• Changes in critical areas likely detrimental• DNA that encodes rRNA is highly conserved among

bacteria of common ancestry

Phylogenetic trees are based on rRNA sequences

transcription translation

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Translation Initiation

3’ 5’ A N U N

UCCUCCA5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’

3’ end of16S rRNA

mRNA

Shine-Delgarnosequence

InitiationCodon

Ribosome

Ribosome Binding Site

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Sensitivity of rRNA

rRNA critical for protein synthesis• binds initiation site (Ribosome binding site,

Shine-Delgarno sequence) in mRNA• must have 2o structure (base pairs with self)• Changes in critical areas likely detrimental• DNA that encodes rRNA is highly

conserved among bacteria of commonancestry

Phylogentic trees are based on rRNA sequences

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http://asiago.stanford.edu/RelmanLab/supplements/Nikkari_EID_8/nikkari2002.html

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Sensitivity of rRNA

rRNA critical for protein synthesis• binds initiation site (Ribosome binding site,

Shine-Delgarno sequence) in mRNA• must have 2o structure (base pairs with self)• Changes in critical areas likely detrimental• DNA that encodes rRNA is highly

conserved among bacteria of commonancestry

Phylogentic trees are based on rRNA sequences

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