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Fundamentals II: Introduction to Bacteriology and Bacterial Structure Janet Yother, Ph.D. Department of Microbiology 4-9531.

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Presentation on theme: "Fundamentals II: Introduction to Bacteriology and Bacterial Structure Janet Yother, Ph.D. Department of Microbiology 4-9531."— Presentation transcript:

1 Fundamentals II: Introduction to Bacteriology and Bacterial Structure Janet Yother, Ph.D. Department of Microbiology

2 Learning Objectives Fundamental properties of prokaryotes Basic structures of bacteria Gram-positive vs Gram-negative bacteria

3 Domains (Kingdoms) Based on evolutionary relationships Eukaryote (Plants, Animals, Protists, Fungi) Eubacteria (Eubacteria) Archaea (Archaea)

4

5 Distinctive Features of Prokaryotic and Eukaryotic Cells Cell componentProkaryotesEukaryotes NucleusNo membrane; single, (usually) circular chromosome Membrane-bound; a number of individual chromosomes Extrachromosomal DNAOften present (plasmids, phage)In organelles Organelles in CytoplasmNoneMitochondria (and chloroplasts in photosynthetic organisms) Cytoplasmic MembraneRespiration, secretion, macromolecular synthesis Lacks functions of prokaryotic membrane Cell WallPeptidoglycan (absent in Mycoplasma) No peptidoglycan (cellulose, chitin in some) SterolsAbsent (except in Mycoplasma)Usually present Ribosomes70S (50S + 30S)80S (60S + 40S)

6 Bacterial Nomenclature KingdomProkaryotae DivisionGracilicutes ClassScotobacteria Subclass OrderSpirochaetales FamilySpirochaetaceae Tribe GenusBorrelia SpeciesBorrelia burgdorferi

7 BACTERIA

8 Prokaryotic Cell Morphology

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10 BACTERIAL CELL 50% protein 20% nucleic acids (10x more RNA than DNA) 10% polysaccharides 10% lipids

11 ori C 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 (s maller = more dependent on host/environment) Up to 1 mm in length; supercoiled Contained in nucleoid * ~1 kb/gene

12 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 cereus Light Microscopy 2500x Feulgen strain Escherichia coli Electron microscopy Jawetz Med Micro 25e

13 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

14 Bacterial Structure

15 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)

16 Cell Wall Shape Barrier (osmotic resistance) Comprised of highly crosslinked peptidoglycan Affected by antibacterials (e.g,  -lactam antibiotics, lysozyme) Basis for gram-stain

17 Peptidoglycan Backbone of N-acetyl glucosamine and N- acetyl muramic acid Cross-linked by peptide bridges at MurNAc

18 Peptidoglycan

19 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 vary between/among Gm+ and Gm-. This = Gm+.  -lactams resemble TP substrates, block crosslinking of growing chain Transglycosylases (TG) link

20  -lactams and Peptidoglycan Crosslinking Transpeptidase [GlcNAc-MurNAc] n L-ala D-glu L-lys D-ala non-crosslinked peptidoglycan CH 3 HC CH CH 3 C HN O NH HOOC Terminal D-ala-D-ala  -lactam ring CH 2 C O NH CH (CH 3 ) 2 HOOC C N O HC C S Benzylpenicillin (penicillin G) R

21 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)

22 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)

23 Gram-positives Cytoplasmic Membrane Cell wall Lipoteichoic acid Teichoic acid Proteins

24 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) R1 = H or Ala; R2 = H or Glc

25 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

26 Gram-negatives Cytoplasmic membrane Cell Wall Outer membrane Lipopolysaccharide Proteins

27 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

28 Lipopolysaccharide (LPS) Endotoxin - toxic shock; fever. leukopenia, hypotension, acidosis, DIC, death (OM)-Lipid A --- core polysaccharide --- O Ag toxic propertiesvaries with species polysaccharide varies with strain sugars/repeat Up to 25 repeats serotyping MMLM HM

29 Gram-negative Surface (Cytoplasmic Membrane)

30 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

31 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


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