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Sophomore Dental andOptometry Microbiology:
Bacterial Structure andPhysiology
Janet Yother, Ph.D.Department of Microbiology
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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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