INTRODUCTION TO BACTERIAL TAXONOMY Dr. El-Safey Mohamed El-Safey Introduction Since the days of Koch and Pasteur bacteria were mainly classified according to morphological, physiological and biochemical criteria. These properties are usually counted equally and bacteria are grouped together as to how many of these features they have in common. This classical approach is also known as numerical taxonomy and the most widely used textbook in bacterial classification Bergey's Manual of Determinative Bacteriology depends heavily on numerical taxonomy. More recently however it has become apparent that many of these original criteria have no bearing at all in defining a particular taxonomic group; this includes such important properties such as cell shape (apart from Spirillae), photosynthesis and to a lesser extent Gram-stain. Due to these shortcomings and the paucity in diversity within alternative techniques scientists were for a long time interested in the development of more useful classification tools. Recent developments in molecular genetics and gene cloning provided exactly these tools giving rise to the new era of molecular taxonomy where a wealth of data can readily be obtained from any microorganisms. 2 BACTERIAL STRUCTURE, CLASSIFICATION, AND PHYSIOLOGY BACTERIA • Prokaryotes - lack a nuclear membrane (unlike eukaryotes) • Single-celled • Reproduce by simple division, i.e. binary fission • Cells - small, ~1 µm (mycoplasmas as small as 0.2 µm; bacillus as large as 10 µm) • Chromosome - single, circular, double-stranded DNA (borrelia - linear); up to 1 mm in length; 600 to 4500 kb in size. Smaller = more dependent on host/environment; larger can synthesize more of own constituents. • Extrachromosomal DNA - plasmids may be present. In cytoplasm, replicate independent of chromosome. Usually circular, sometimes linear (Borrelia). Few to several hundred kb. • Most are free-living, a few (rickettsiae, chlamydiae) are obligate intracellular parasites THE PLACE OF BACTERIA IN THE GRAND SCHEME OF THINGS 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, all pathogens, most well-studied. • Archaebacteria - primitive bacteria. Evolutionarily separated from eubacteria. Differences in cell walls (lack PG), membranes (ether- rather than ester-linked lipids), ribosome components and metabolism. Share some features with euk (introns, histones, etc). Groups of archaebacteria: Methanogens - produce methane 3 Halophiles - grow in high salt Thermophiles - grow at high temp Based on evolutionary relationships (more later), 3 domains (kingdoms) suggested: Archaebacteria Eubacteria Eukaryotes BACTERIAL STRUCTURE 1. nucleoid (nuclear or chromatin body) • DNA (60%; 2-3% dry wt of cell); RNA (30%); protein (10%) • Does not have a nuclear membrane • Haploid chromosome in cytoplasm • 1 to 4 nuclear bodies/cell, number depends on growth rate example: E. coli at 37 o C - doubling time = 20 min. - 4 copies; with dt > 60 min - 1 copy (more later) • No histones, but ~6 chromosome-associated basic proteins involved in determining chromosomal structure • Polyamines, e.g., spermidine and putrescine, neutralize negative charges on phosphates • Size of E. coli chromosome = 3 x 10 9 daltons = 4.7 x 10 6 bp • Roughly 4000 +/- 1000 genes • Circular and double stranded (borrelia - linear) • 1.6 mm - supercoiled (underwound) to fit (occupies 10% of cell volume) • Smaller bacteria like Rickettsia and Chlamydia have chromosomes less than one- third the size of that of E. coli. Smaller chromosome, more dependent on host/environment. 2. cytoplasmic membrane (CM) (inner membrane, IM in gram -) • lipid bilayer with embedded proteins, little carbohydrate. • osmotic barrier, active transport of ions and metabolites, electron transport, oxidative phosphorylation,and photosynthesis (in some bacteria) • 7.5 nm thick • antibacterials: detergents, polymyxins, ionophores disrupt membrane 3. mesosomes - cytoplasmic membrane-associated organelle (more easily seen in gram-positive bacteria); an irregular invagination in the cytoplasmic membrane. 4 • Note: bacteria don't have the membranous organelles of eukaryotes, e.g., endoplasmic reticulum, mitochondria, golgi, etc. • May be attached to the nucleoid • Possible role in replication and cell division (septal meso), maybe in protein export (lateral meso) 4. cell wall (CW) (murein) - shape, barrier (osmotic resistance - CW removed by enzymes that degrade, cells will lyse in water, serum. Protoplasts = wall-less, osmotically sensitive (gm +); spheroplasts = same with some envelope component retained = gm -) • Peptidoglycan (PG) - highly crosslinked layers of NAM-NAG joined peptides, crosslinking in peptide. Certain antibiotics ( -lactams, penicillins) prevent crosslinks, lysozyme degrades. • DAP (diaminopimelic acid) in PG is unique to bacteria • Cell wall thickness is basis for gram stain: • property of cell wall to retain a basic dye (Gram's crystal violet/KI/acetone/wash/safranin) • those that don't retain crystal violet are counterstained with safranin (red) • thick cell walls retain (= gram +); thin cell walls don't (= gram -). Something else may be involved. • Exceptions to gram-positive/gram-negative staining: • mycoplasmas - no cell wall. Triple-layered membrane, gets sterols from host. • mycobacteria - lipid interferes with strain; use an acid-fast stain (retain carbol fuschin when decolorized with HCl in alcohol). • both are related to gram-positives based on genetic analyses (rRNA sequence). In gm + only: • Teichoic acids (Wall TA = WTA) - oligosaccharides (roughly 30 ribitol residues with phosphodiester links). Covalently linked to PG. 50% of dry weight of CW; 10% of dry weight of cell. • Lipoteichoic acids, LTA (polyglycerol phosphates) attached to the cytoplasmic membrane. May be same or different structure as teichoic. • Both, bind ions, important in membrane integrity, adherence 5. outer membrane (OM) - only in gram-negative bacteria • bilayer composed of phospholipids, proteins (4 - 5 major, 10 - 50 minor), and LPS 5 • LPS = Lipid A + core polysaccharides + O Ag = Endotoxin (Lipid A is responsible for toxic effects –endotoxic shock: fever, hypotension, impaired organ function, acidosis, dissem. intravascular coag., death). • contains lipoprotein (lipid/protein molecule covalently attached to the cell wall) • second permeability barrier, excluding molecules >600 daltons, e.g., many antibiotics • contains porins (trimeric protein pores; channels for low MW water soluble substances, phage receptors) (Note that in gram-positive bacteria, molecules as large as 10 5 daltons can pass through the cell wall.) Bayer's junctions (adhesion zones) - points of connection between IM and OM. • outside = phage attachment/injection site, complement-mediated lysis • inside = growth zones, sites for translocation of OM proteins, polysaccharides, LPS, pili, flagella 6. periplasmic space - space between IM and OM (or CW in gram-positive) • very small or nonexistent in gram-positive lactamase-ك contains hydrolytic enzymes like alkaline phosphatase and • • contains amino acid and sugar binding (transport) proteins • contains chemoreceptors • contents can be released by osmotic shock - rapid dilution of hypertonic (0.5 M sucrose) cell suspensions with H 2 0, after treatment with EDTA 7. "Optional" structures (Gm + and Gm -) capsule • varying thickness • polysaccharide or protein • antiphagocytosis, attachment • structurally, antigenically variable within a species • tightly associated with cells; if it can be washed off easily, it's called slime flagella (singular = flagellum) - motility, chemotaxis, virulence. FIGURE 5 • 3 parts: basal body and hook in cell envelope plus extended filament • filament: long (7-15 µm) and thin (20 nm diam.) (can't see with light microscope - need to stain) composed primarily of a single, self-aggregating protein called flagellin • basal body and hook: motor for energy-dependent rotation of the the filament • one to several hundred per cell • peritrichous - all around cell 6 • polar - at one end of the cell • bipolar - both ends pili (fimbriae) (singular = pilus) - hair-like structures composed of single, self- aggregating protein (pilin) • shorter, thinner, and straighter than flagella (several µ long, 7.5 -10 nm thick) • types: • common pili - peritrichous; attachment • F (sex) pilus - single; gene transfer (conjugation); gram-negative only granular inclusions or inclusion bodies - -storage polymers; often visualized by hydroxybutyrate-ك-staining. Glycogen, polyphosphate, poly endospores (spores) - dehydrated cells that are resistant to heat (difficult to kill); more later toxins - excreted (either actively or by cell lysis) by bacterium, act on host cells. Virulence. Examples: • cholera toxin (Vibrio cholerae) - B subunit (pentamer) binds host cells (GM1); A subunit has toxic activity (increases adenlyate cyclase activity, cAMP concentrates, electrolytes are secreted, Na and Cl absorption in small bowel impaired, fluid and electrolytes lost). • tetanus toxin - (Clostridium tetani) - reaches peripheral nervous system, blocks release of neurotransmitter, acetylcholine, from nerve terminals at neuromuscular junctions. ADP-ribosylating activity. Released at bacterial cell death. Bacteriophage- encoded. enzymes - excreted, act on extracellular polymers, host cells; hyaluronidases, proteases, DNases. BACTERIAL TAXONOMY Classification