Fundamentals II:Introduction to Bacteriology and

Bacterial Structure

Janet Yother, Ph.D.Department of Microbiology

jyother@uab.edu4-9531

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

BACTERIA

Prokaryotic Cell Morphology

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

Bacterial Structure

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

Peptidoglycan

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

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+.

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

Transglycosylases (TG) link

b-lactams and Peptidoglycan Crosslinking

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

b-lactamring

CH2

C ONHCHCH

(CH3)2HOOC

CN

O

HC C S

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-positives

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

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

HNAcO

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

OCH2

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

• Cytoplasmic membrane

• Cell Wall• Outer membrane• Lipopolysaccharide• Proteins

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 Agtoxic properties varies with species polysaccharide

varies with strain3 - 4 sugars/repeatUp to 25 repeatsserotyping

MM LM

HM HM

Gram-negative Surface

(Cytoplasmic Membrane)

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