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Introduction to Bacteriology Joan Olson - HSC 2090 jolson@hsc.wvu.edu

Introduction to Bacteriology Joan Olson - HSC 2090 jolson@hsc.wvu.edu. Journal article presentation: Introduction - background of studies Results - linked to figures experimental approach / techniques conclusion from figure Summary / conclusion - significance of findings of paper.

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Introduction to Bacteriology Joan Olson - HSC 2090 jolson@hsc.wvu.edu

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  1. Introduction to BacteriologyJoan Olson - HSC 2090jolson@hsc.wvu.edu

  2. Journal article presentation: Introduction - background of studies Results - linked to figures experimental approach / techniques conclusion from figure Summary / conclusion - significance of findings of paper

  3. Objectives of Microbial Pathogenesis section: - Introduction to the world of bacteria - Examine strategies of bacterial pathogenesis using model organisms toxins intracellular pathogens bacterial secretion emerging pathogens microbes and cancer • Become aware of the relationship and inter-relationship • of bacterial and eukaryotic processes • Highlight use of bacterial systems to study and manipulate • eukaryotic cells • www.bact.wisc.edu/bact330/bact330homepage Ken Todar • Text:Bacterial Pathogenesis - A Molecular Approach (2nd Edition) • Abigail Salyers and Dixie Whitt

  4. Inter-relationship between bacteria and eukaryotes JL Gould, CG Gould (1989) Life at the Edge

  5. Co-existence / co-dependence Eukaryotes - develop / require symbiotic relationship with bacteria energy nutrient digestion microbial competition (normal flora) Bacteria - develop mechanisms to utilize / modulate eukaryotes direct and indirect mechanisms Pathogenicity is rare

  6. What is the world of bacteria?

  7. Evolutionary relationship between prokaryotes and eukaryotes chloroplasts (cyanobacteria) mitochondria (proteobacteria) CM rRNA rRNA rRNA CW CM CM CW Archaea halophiles methanogens thermophiles thermoplasma CW OM (www.science.mcmaster.ca/ biochem/faculty/gupta…)

  8. Features of prokaryotic and eukaryotic cells Component Prokaryotes Eukaryotes Nucleus DNA not membrane bound DNA membrane bound ds DNA ds DNA usually one circular chromosome multiple, linear chromosomes Other DNA plasmids often present in organelles Organelles none mitochondria & chloroplasts Cytoplasmic respiration, ATP synthesis transport & secretion membrane transport & secretion Cell wall rigid layer of peptidoglycan no peptidoglycan (except Mycoplasma) Sterols absent (except Mycoplasma) present Ribosomes 70S 80S

  9. Microbes are small, abundant, diverse and everywhere (“micro” + “bios”) a typical bacterial cell is ~ 1 mm (from 0.4-100 mm)bacteria can be seen (barely) using the light microscope bacteria are the most numerous (free-living) organism on earth extremely diverse only a small fraction of all species has been cultivated or studied only a small fraction of species interact with humans

  10. History of microbiology: Antoj van Leeuwenhoek 1623-1723: (Frobisher et al., Fundamentals of Microbiology, 1974)

  11. Van Leeuwenhoek’s microscope van Leeuwenhoek - had sharp eyesight and unfailing curiosity

  12. Pasteur’s prinicples: Every alteration, either of beer or of wine depends on the development in it of microorganisms which are ferments or “diseases” of beer or wine. These “germs or ferments” are brought by air, by the ingredient, or by the apparatus used in breweries. Whenever beer or wine contains no livingmicroorganisms it remains unchanged. Louis Pasteur 1822-1895

  13. Hans Christian Gram 1843-1938 Gram (differential) stain - 1884 Fix organisms to slide with heat and stain with basic dye (crystal violet ) - then rinse Apply iodine solution to form dye-I2 complex - then rinse Wash with ethyl alcohol or acetone Gram-positive bacteria remain blue Apply red counterstain (safranin) Gram-negative bacteria stained red

  14. How are bacteria characterized?

  15. Escherichia coil Campylobacter jejuni Borrelia burgdorferi (Lyme disease) Vibrio cholerae (UCLA Department of Epidemiology website) www.nature.com/genomics / papers/c_jejuni.html (© 1998 American Museum of Natural History). (mysite.verizon.net/ res6ogjw/) Structure ~ Shape Arrangement

  16. Staphylococcus aureus (www.buddycom.com/bacteria/ gnr/acinetc1259.jpg) (www.silvermedicine.org/ colloidalsilverstudyte…) Pseudomonas aeruginosa Spirillum minus (www.geocities.com/ CapeCanaveral/3504/gallery.htm) (www.geocities.com/ CapeCanaveral/3504/gallery.htm) Gram stain ~ Gram-positiveGram-negative Clostridium botulinum

  17. [M. Battaglia, D. Pozzi, S. Grimaldi, T. Parasassi. 1994 Biotechnic & Histochemistry 69:152-156]. (non-Gram staining bacteria) Mycoplasma- infected cell culture Hoechst 33258 fluorochrome staining Mycobacterium tuberculosis acid fast stain (medinfo.ufl.edu/year2/mmid/bms5300/bugs/mycotubr.html) lacks a cell wall thick, waxy, lipid rich cell wall - resistant to staining

  18. Metabolism ~ Source of energy photosynthetic / chemosynthetic -obtain energy from sunlight or from oxidizing inorganic compounds(autotroph) (chemo)heterotrophs -require organic compounds for energy (all pathogens are heterotrophs) saprophyte - feeds on decaying or dead organic material Cellular respiration aerobe -use oxygen for respiration - final electron acceptor is oxygen anaerobe -use fermentation for respiration - final electron acceptor is organic molecules facultative anaerobe -can use oxygen or fermentation for respiration

  19. Bacterial growth curve Bacteria diluted into liquid medium / growth monitored using a spectrophotometer lag phase - initial adjustment to medium log / expodential phase - rapid exponential growth (e.g. E. coli generation time 20 min = 1000-fold increase in 3.5 hours) stationary phase - limiting nutrients / toxicity eventually growth stops, sometimes see loss of viability with time

  20. Carl Woese - 1977 Universal phylogenic tree - based on SSU rRNA sequences Molecular composition ~

  21. Comparative genomes genes 483 4377 5570 6,000 35,000 -100,000 size (kb) 500 4600 6300 12,495 3,300,000 Prokaryote mycoplasma Escherichia coli Pseudomonas Eukaryote yeast humans Genomes ~ • prokaryotic genome - • most bacteria have a single large circular chromosome • • all bacteria are haploid

  22. Completed microbial genomes 1995-2004 Organism Size Acinetobacter sp. ADP13.60 Aeropyrum pernix K11.67 Agrobacterium tumefaciens str. C585.67 Agrobacterium tumefaciens str. C585.67 Aquifex aeolicus VF51.59 Archaeoglobus fulgidus DSM 43042.18 Bacillus anthracis str. A20125.37 Bacillus anthracis str. Ames5.23 Bacillus anthracis str. Ames 05815.50 Bacillus anthracis str. Sterne5.23 Bacillus cereus ATCC 109875.43 Bacillus cereus ATCC 145795.43 Bacillus cereus ZK5.30 Bacillus halodurans C-1254.20 Bacillus licheniformis DSM 134.22 Bacillus licheniformis DSM 134.22 Bacillus subtilis subsp. subtilis str. 1684.21 Bacillus thuringiensis serovar konkukian str. 97-27 5.24 Bacteroides fragilis YCH465.31 Bacteroides thetaiotaomicron VPI-54826.29 Bartonella henselae str. Houston-11.93 Bartonella quintana str. Toulose1.58 Bdellovibrio bacteriovorus HD1003.78 Bifidobacterium longum NCC27052.26 Bordetella bronchiseptica RB505.34 Bordetella parapertussis 128224.77 Bordetella pertussis Tohama I4.09 Borrelia burgdorferi B311.52 Borrelia garinii PB10.99 Bradyrhizobium japonicum USDA 1109.11 Brucella melitensis 16M3.29 Brucella suis 13303.32 Buchnera aphidicola str. APS (Acyrthosiphon pisum 0.66 Buchnera aphidicola str. Bp (Baizongia pistaciae)0.62 Buchnera aphidicola str. Sg (Schizaphis graminum)0.64 Burkholderia mallei ATCC 233445.84 Burkholderia pseudomallei K962437.25 Campylobacter jejuni subsp. jejuni NCTC 111681.64 Candidatus Blochmannia floridanus0.71 Caulobacter crescentus CB154.02 Chlamydia muridarum1.08 Chlamydia trachomatis D/UW-3/CX1.04 Chlamydophila caviae GPIC1.18 Chlamydophila pneumoniae AR391.23 Chlamydophila pneumoniae CWL0291.23 Chlamydophila pneumoniae J1381.23 Chlamydophila pneumoniae TW-1831.23 Chlorobium tepidum TLS2.15 Chromobacterium violaceum ATCC 12472 4.75 Clostridium acetobutylicum ATCC 8244.13 Clostridium perfringens str. 133.09 Clostridium tetani E882.87 Corynebacterium diphtheriae NCTC 131292.49 Corynebacterium efficiens YS-3143.15 Corynebacterium glutamicum ATCC 130323.31 Coxiella burnetii RSA 493 [ 2.03 Deinococcus radiodurans R13.28 Desulfotalea psychrophila LSv543.66 Desulfovibrio vulgaris subsp. vulgaris str. Hildenboro 3.77 Enterococcus faecalis V5833.36 Erwinia carotovora subsp. atroseptica SCRI10435.06 Escherichia coli CFT0735.23 Escherichia coli K124.64 Escherichia coli O157:H75.50 Escherichia coli O157:H7 EDL9335.53 Fusobacterium nucleatum subsp. nucleatum ATCC 2 2.17 Geobacter sulfurreducens PCA3.81 Gloeobacter violaceus PCC 74214.66 Haemophilus ducreyi 35000HP1.70 Haemophilus influenzae Rd KW201.83 + Haloarcula marismortui ATCC 430494.27 Halobacterium sp. NRC-12.57 Helicobacter hepaticus ATCC 514491.80 Helicobacter pylori 26695 1.67 Helicobacter pylori J991.64 Lactobacillus johnsonii NCC 5331.99 Lactobacillus plantarum WCFS13.35 Lactococcus lactis subsp. lactis Il14032.37 Legionella pneumophila str. Lens3.41 Legionella pneumophila str. Paris3.64 Legionella pneumophila subsp. pneumophila str. Phila3.40 Leifsonia xyli subsp. xyli str. CTCB072.58 Leptospira interrogans serovar Copenhageni str. Fiocr4.63 Leptospira interrogans serovar lai str. 566014.69 Listeria innocua Clip11262 3.09 Listeria monocytogenes EGD-e2.94 Listeria monocytogenes str. 4b F23652.91 Mannheimia succiniciproducens MBEL55E2.31 Mesoplasma florum L10.79 Mesorhizobium loti MAFF3030997.60 Methanocaldococcus jannaschii DSM 26611.74 Methanococcus maripaludis S21.66 Methanopyrus kandleri AV191.69 Methanosarcina acetivorans C2A5.75 Methanosarcina mazei Goe14.10 Methanothermobacter thermautotrophicus str. Delta H1.75 Methylococcus capsulatus str. Bath3.30 Mycobacterium avium subsp. paratuberculosis str. K1 4.83 Mycobacterium bovis AF2122/974.35 Mycobacterium leprae TN3.27 Mycobacterium tuberculosis CDC15514.40 Mycobacterium tuberculosis H37Rv4.41 Mycoplasma gallisepticum R1.00 Mycoplasma genitalium G-370.58 Mycoplasma hyopneumoniae 2320.89 Mycoplasma mobile 163K0.78 Mycoplasma mycoides subsp. mycoides SC str. PG11.21 Mycoplasma penetrans HF-21.36 Mycoplasma pneumoniae M1290.82 Mycoplasma pulmonis UAB CTIP0.96 Nanoarchaeum equitans Kin4-M0.49 Neisseria meningitidis MC582.27 Neisseria meningitidis Z24912.18 Nitrosomonas europaea ATCC 197182.81 Nocardia farcinica IFM 101526.29 Nostoc sp. PCC 71207.21 Oceanobacillus iheyensis HTE8313.63 Onion yellows phytoplasma OY-M0.86 Parachlamydia sp. UWE252.41 Pasteurella multocida Pm702.26 Photobacterium profundum SS96.40 Photorhabdus luminescens subsp. laumondii TTO15.69 Picrophilus torridus DSM 97901.55 Porphyromonas gingivalis W832.34 Prochlorococcus marinus str. MIT 93132.41 Prochlorococcus marinus subsp. marinus str. CCMP1 1.75 Prochlorococcus marinus subsp. pastoris str. CCMP1 1.66 Propionibacterium acnes KPA1712022.56 Pseudomonas aeruginosa PAO16.26 Pseudomonas putida KT24406.18 Pseudomonas syringae pv. tomato str. DC30006.54 Pyrobaculum aerophilum str. IM22.22 Pyrococcus abyssi GE51.77 Pyrococcus furiosus DSM 36381.91 Pyrococcus horikoshii OT3 1.74 Ralstonia solanacearum GMI10005.81 Rhodopseudomonas palustris CGA0095.46 Rickettsia conorii str. Malish 71.27 Rickettsia prowazekii str. Madrid E1.11 Rickettsia typhi str. wilmington1.11 Salmonella enterica subsp. enterica serovar Typhi Ty 4.79 Salmonella enterica subsp. enterica serovar Typhi str.5.13 Salmonella typhimurium LT24.95 Shewanella oneidensis MR-15.13 Shigella flexneri 2a str. 2457T4.60 Shigella flexneri 2a str. 3014.83 Sinorhizobium meliloti 10216.69 Staphylococcus aureus subsp. aureus MRSA2522.90 Staphylococcus aureus subsp. aureus MSSA4762.82 Staphylococcus aureus subsp. aureus MW22.82 Staphylococcus aureus subsp. aureus Mu502.90 Staphylococcus aureus subsp. aureus N3152.84 Staphylococcus epidermidis ATCC 122282.50 Streptococcus agalactiae 2603V/R2.16 Streptococcus agalactiae NEM3162.21 Streptococcus mutans UA1592.03 Streptococcus pneumoniae R62.04 Streptococcus pneumoniae TIGR42.16 Streptococcus pyogenes M1 GAS1.85 Streptococcus pyogenes MGAS103941.90 Streptococcus pyogenes MGAS3151.90 Streptococcus pyogenes MGAS82321.90 Streptococcus pyogenes SSI-11.89 Streptomyces avermitilis MA-46809.12 Streptomyces coelicolor A3(2)9.05 Sulfolobus solfataricus P22.99 Sulfolobus tokodaii str. 72.69 Symbiobacterium thermophilum IAM 148633.57 Synechococcus sp. WH 81022.43 Synechocystis sp. PCC 68033.57 Thermoanaerobacter tengcongensis2.69 Thermoplasma acidophilum DSM 17281.56 Thermoplasma volcanium GSS11.58 Thermosynechococcus elongatus BP-12.59 Thermotoga maritima MSB81.86 Thermus thermophilus HB272.13 Treponema denticola ATCC 354052.84 Treponema pallidum subsp. pallidum str. Nichols1.14 Tropheryma whipplei TW08/270.93 Tropheryma whipplei str. Twist0.93 Ureaplasma parvum serovar 3 str. ATCC 7009700.75 Vibrio cholerae O1 biovar eltor str. N169614.03 Vibrio parahaemolyticus RIMD 22106335.17 Vibrio vulnificus CMCP65.13 Vibrio vulnificus YJ0165.26 Wigglesworthia glossinidia endosymbiont of Glossina 0.70 Wolbachia endosymbiont of Drosophila melanogaster1.27 Wolinella succinogenes DSM 17402.11 Xanthomonas axonopodis pv. citri str. 3065.27 Xanthomonas campestris pv. campestris str. ATCC 335.08 Xylella fastidiosa 9a5c2.73 Xylella fastidiosa Temecula12.52 Yersinia pestis KIM4.60 Yersinia pestis biovar Mediaevails str. 910014.80 Yersinia pseudotuberculosis IP 329534.84

  23. Pseudomonas aeruginosa genome: • regulatory genes • genes involved in transport • catabolism, efflux of organics • Clonal genotypes, reflect large deletions or insertions Insertion of >80-100 kb gene islands or plasmids (in tRNA loci) (Stover et al, 2000; Kiewitz et al, 2000; Kiewitz 2002) Genome characteristics ~

  24. mechanisms for gene transfer: transformation -transfer of (naked) DNA through cell membrane - occurs without cell-cell contact and is nuclease-sensitive in nature - DNA is generally released by lysis / taken-up by same species in laboratory - DNA is transferred using high voltage - electroporation Bacterial genome evolution ~ high frequency of genetic exchange between bacteria conjugation - one-way transfer of DNA from donor to recipient bacteria, requires direct cell-cell contact, usually plasmid-mediated conjugation requires a plasmid carrying conjugation genes (F or R plasmid) plasmids encode proteins that function in DNA transfer and pilus formation plays a major role in transfer of drug resistance transduction -DNA carried into cell by a bacterial virus (phage) involved in transfer of virulence factors between bacteria specialized transduction - host cell carries phage in dormant statelysogen only a few viral genes expressed (e.g. toxin genes)

  25. mechanisms for gene incorporation: recombination - physical exchange between two DNA molecules homologous recombination - requires extensive homology non-homologous recombination - occurs between unrelated DNA requires transposon / insertion sequences Bacterial genomic terminology ~ genotype -the state of the genes in an organism mutation -anychange in the sequence of a gene phenotype -observable properties (wild type / mutant phenotype) prototroph -wild type phenotype(relates to original parental reference strain) auxotroph - mutant that cannot make an essential small molecule (amino acid, nucleic acid base, vitamin) Genome evolution ~ bacterial gene transfer and recombination technology ~ integral to all genetic manipulations ~ => mutant organism

  26. Anatomy of a bacterium ~

  27. Plasmid extranuclear genetic element replicates autonomously encodes non-essential genes (e.g. antibiotic resistance, virulence factors) 70S rRNA short, thin, straight projections used for adhesion / specialized pili used for DNA transfer (conjugation) rotating helical filament used for swimming/chemotaxis

  28. Anatomy of Escherichia coli (Dennis Kunkel Microscopy, 2001)

  29. Cell envelope Gram-negative vs. Gram-positive (Schaechter, The Desk Encyclopedia of Microbiology, 2004)

  30. Gram-negative and Gram-positive cell envelope

  31. D-ala D-ala L-DAP/ L-lysine D-glu L-ala Peptidoglycan cell wall (murein) (peptide + glycan cross-linked mesh) peptide N-acetylmuramic acid N-acetylglucosamine glycan cross-link

  32. L V * * * * P * * P * isopeptide bond - teminal carboxyl bond to non-alpha amine penicillin -D-ala-D-ala analogue, inhibits transpeptidase cross-linking vancomycin - inhibits glycosyl polymerization lysozyme -cleaves b-1,4 NAM-NAG bond Peptidoglycan properties Gram-negative Gram-positive NAG NAM NAG NAM D-ala L-Lys D-glu L-ala D-ala G-G-G-G-G L-Lys D-ala L-DAP (diaminopimelic acid) D-ala L-DAP D-glu L-ala LD - DD peptide bonds -render peptides resistant to peptidases bacitracin - blocks peptidoglycan precursor synthesis

  33. outer membrane structure - additional permeability barrier outer leaflet is lipopolysaccharide inner leaflet is typical phospholipid monolayer periplasm - (protein assembly) cytoplasmic membrane - typical phospholipid bilayer - functions in energy production, transport,secretion, signaling porin protein trimer channel - allows nutrient transfer past outer membrane of Gram-negative bacteria channel size regulates access - e.g. vancomycin and bacitracin too large to pass through channel Gram-negative cell envelope

  34. Lipopolysaccharide (LPS) - endotoxin O-antigen -long chain of sugar repeats (highly variable) - target of antibody response & generation of antibody-serotyping reagents (E. coliO157:H7) core oligosaccharide - IC: composed of Kdo and Hep sugars OC: glycose lipid A -active component of LPS - essential for bacterial growth - Lipid A + core oligosaccharide similar for all Gram-negative bacteria LPS important in immune function - stimulates macrophages => IL-1 + TNF - low amounts cause fever - high amounts cause septic shock / inflammation - experimental immunology tool - activates B-cells in mice (not humans)

  35. teichoic acid -polymers of alcohol phosphate (ribitol or glycerol) - decorated with amino acids or sugars - highly variable and antigenic -important for attachment to target cells lipoteichoic acid - teichoic acid attached to glycolipid surface proteins - adhesin used to adhere to particular cell type (e.g. M-protein of GAS) Gram-positive cell envelope (www.cat.cc.md.us/courses/bio141/ lecguide/unit1/bacpath/ gpcw/gpcw.html)

  36. Pathogen-associated molecular patterns (PAMPS) O-antigen Core-PS Lipid A lipoteichoic acid LPS / endotoxin

  37. Electron micrograph of a bacterium showing a large quantity of surrounding capsular material (EPS). (www.activatedsludge.info/ resources/visbulk.asp) Capsule Slime - associated with both Gram-positive and Gram-negative bacteria - protects against phagocytosis & drying - metabolic commitment - shift to production of large amounts of polysaccharides (variable sequence among different species) - capsule production ‘smooth’ / no capsule production ‘rough’

  38. Sporulation ~ Under conditions of nutrient limitation (C or N) some Gram-positive bacteria form spores (Clostridia, Bacillus) Spores - highly dehydrated / do not metabolize - can survive for many years (anthrax > 1000 y) - resistant to heat, drying, freezing, ionizing radiation - best killed with heat and water (i.e. steam)

  39. Types of bacterial-host interactions parasitic - an association where one organism gains benefit from another without compensation (may cause harm) symbiotic -an association between organisms from which each derives benefit commensal -an association with an organism that causes no harm(normal flora)

  40. Normal flora ~1013 mammalian cells in the body ~1014 bacteria living on / in each of us (~ 1000 different species) most of these bacteria are commensal also referred to as - normal microflora or ‘indigenous microbiota’ normal flora important for human health digestive functions supply vitamins inhibit infections - compete with pathogenic species for colonization

  41. Normal flora (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002)

  42. Normal flora microflora communities are extremely complex enormous variation in microflora from - individual to individual & site to site little is known about the interaction of microflora with mammalian cells and tissues bacteriological research has focused on disease

  43. Concepts • evolutionary relationship / differences between prokaryotes and eukaryotes • bacterial structure / metabolism / molecular biology / genomics ~ • (importance of bacterial genetics to biotechnology and recombinant DNA • technology) • anatomy of a bacterium ~ differences between Gram-positive and Gram- • negative bacteria • understanding of the abundance, diversity and distribution of bacteria in • nature • types of bacterial-host interactions ~ importance / function of normal flora

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