fundamentals ii: introduction to bacteriology and bacterial structure janet yother, ph.d. department...
TRANSCRIPT
Fundamentals II:Introduction to Bacteriology and
Bacterial Structure
Janet Yother, Ph.D.
Department of Microbiology
4-9531
Learning Objectives
• Fundamental properties of prokaryotes• Basic structures of bacteria• Gram-positive vs Gram-negative bacteria
Domains (Kingdoms)Based on evolutionary relationships
• Eukaryote (Plants, Animals, Protists, Fungi)• Eubacteria (Eubacteria)• Archaea (Archaea)
Distinctive Features of Prokaryotic and Eukaryotic Cells
Cell component Prokaryotes Eukaryotes
Nucleus No membrane; single, (usually) circular chromosome
Membrane-bound; a number of individual chromosomes
Extrachromosomal DNA
Often present (plasmids, phage) In organelles
Organelles in Cytoplasm None Mitochondria (and chloroplasts in photosynthetic organisms)
Cytoplasmic Membrane Respiration, secretion, macromolecular synthesis
Lacks functions of prokaryotic membrane
Cell Wall Peptidoglycan (absent in Mycoplasma)
No peptidoglycan (cellulose, chitin in some)
Sterols Absent (except in Mycoplasma) Usually present
Ribosomes 70S (50S + 30S) 80S (60S + 40S)
Bacterial Nomenclature
• Kingdom Prokaryotae• Division Gracilicutes• Class Scotobacteria• Subclass• Order Spirochaetales• Family Spirochaetaceae• Tribe• Genus Borrelia • Species Borrelia
burgdorferi
BACTERIAL CELL
• 50% protein• 20% nucleic acids (10x more RNA
than DNA)• 10% polysaccharides• 10% lipids
oriC
oriC
Bacterial Chromosomes
• Single, circular, double-stranded DNA (exception - borrelia = linear)
• Replication begins at unique point; bidirectional
• Haploid (1 to 4 copies depending on growth rate)
• 600 to >5000 kb* in size (smaller = more dependent on host/environment)
• Up to 1 mm in length; supercoiled• Contained in nucleoid
* ~1 kb/gene
Bacterial Nucleoids• 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 determining chromosomal 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 (during cell
division)Bacillus cereusLight Microscopy 2500xFeulgen strain
Escherichia coliElectron microscopy
Jawetz Med Micro 25e
Extrachromosomal DNA
• Plasmids - Replicate in cytoplasm, independent of chromosome. – Double-stranded DNA; usually circular (borrelia =
linear)– Few to several hundred kb– Few to several hundred copies per cell – Conjugative (F, R), antibiotic resistance,
metabolic, virulence• Bacteriophage - virus;
– replicates in cytoplasm or integrates into into chromosome
– can contribute to virulence
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)
Cell Wall
• Shape• Barrier (osmotic
resistance)• Comprised of highly
crosslinked peptidoglycan• Affected by antibacterials
(e.g, b-lactam antibiotics, lysozyme)
• Basis for gram-stain
Peptidoglycan
• Backbone of N-acetyl glucosamine and N-acetyl muramic acid
• Cross-linked by peptide bridges at MurNAc
http://employees.csbsju.edu/hjakubowski/classes/ch331/cho/peptidoglycan.gif
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.) cleave
Amidases (autolysins, e.g.)cleave
PG structures varybetween/among Gm+and Gm-. This = Gm+.
-lactams resemble TP substrates, block crosslinking of growing chain
Transglycosylases (TG) link
-lactams and Peptidoglycan Crosslinking
Transpeptidase
[GlcNAc-MurNAc]n
L-ala
D-glu
L-lys
D-ala
D-alanon-crosslinked peptidoglycan
CH3HC
CH
CH3
C
HN
ONH
HOOC
Terminal D-ala-D-ala
-lactamring
CH2
C ONHCH
CH
(CH3)2HOOC
C
N
O
HC CS
Benzylpenicillin(penicillin G)
R
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)
Exceptions to gram-positive / gram-negative staining
• Mycoplasmas - no cell wall. • Mycobacteria - lipid interferes with stain
– Detected with acid fast stain (carbol fuschin retained following decolorization with HCl/EtOH)
Both are related to gram-positives, based on genetic analyses (rRNA sequence)
Gram-positive Cell Walls• Thick peptidoglycan (10 to 100 nm)• Wall teichoic acids (WTA) - repeating units of
phosphodiester-linked (negative charge) glycerol or ribitol backbone + side chains (D-ala, glucose).
Covalently linked to PG (MurNAc)
CH2OH
H-C-O-R1 O
H-C-OH P
H-C-O-R2 O-
CH2O
H-O-C-H P H-O-C-H P
O-CH2 O O-CH2 O
CH2O O- CH2O O
-
CH2OH
HNAc
O
O
OH
CH2OH
HNAc
O
OHO
TA Repeat Ribitol-P
(n)
Linkage Unit (LU) Peptidoglycan
(Glycerol-P)2-(N-acetylmannosamine)-GlcNAc-P-----MurNAc-GlcNAc--
OO-
P
O
O
CH2
HNAc
O
peptide
HOO
GlcNAc-
Bacillus subtilus W23
**Glycerol-P TAs also have linkage unit - R groups differ on TA repeat and LU
R1 = H or Ala; R2 = H or Glc
Gram-positive Teichoic Acids• Wall Teichoic Acids (WTA) – covalently linked to PG• Lipoteichoic acids (LTA) – similar to WTA but anchored to
cytoplasmic membrane lipids; phosphodiester-linked (negative charge)
• LTA and WTA • ion binding• charge maintenance
• membrane integrity• adherence• anchor proteins• Cell walls - inflammation
Gram-negatives• Cell Wall
– Thin peptidoglycan (1 layer; 2 nm)– No WTA or LTA
• Periplasmic space - digestive and protective enzymes; transport
• Outer membrane (OM) - blocks entry of large molecules (>800 Da). Not typical lipid bilayer.– Attached to PG by lipoprotein– Lipopolysaccharide (LPS) - forms outer leaflet of OM– OM proteins – transport; porins allow passive
diffusion of low MW hydrophilic compounds (sugars, amino acids)
OmpF
Lipopolysaccharide (LPS)• Endotoxin - toxic shock; fever. leukopenia, hypotension,
acidosis, DIC, death
(OM)-Lipid A --- core polysaccharide --- O Ag
toxic properties varies with species polysaccharidevaries with strain3 - 4 sugars/repeatUp to 25 repeatsserotyping
MM LM
HM HM
Optional Features (Gram +/-)• Capsules - polysaccharide or protein (usually
covalently linked to peptidoglycan)– 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; Clostridium botulinum; Vibrio cholerae
• Enzymes - hyaluronidase, proteases, DNases• Endospores - dehydrated cells; Clostridium, Bacillus
species (gram +)
F-pilus