Bacterial Cell Structure and how it is important

Bacterial cell structure

1. Bacterial Cell Structure, Properties & Metabolism DR. ALEX-WELE M.A. DEPARTMENT OF MEDICAL MICROBIOLOGY & PARASITOLOGY, UPTH.

2. Introduction Schwann and Schleiden : cells basic unit of life Prokaryotes and eukaryotes from microscopy. Our focus: prokaryotic cells. Eubacteria and Archaebacteria

3. Introduction Bacteria are unicellular free living organisms, having both RNA and DNA They are by far the smallest living cells that are capable of performing all essential processes of life, e.g. growth, metabolism and reproduction Although most are capable of independent existence, some are obligate intracellular organisms that depend on their host for survival e.g. Chlamydiae and Rickettsiae

4. Introduction Bacteria are small and simple in structure when compared with eukaryotes, yet they often have characteristic shapes and sizes. Although they have a plasma membrane, which is required by all living cells, bacteria generally lack extensive, complex, internal membrane systems . The cytoplasmic matrix typically contains several constituents that are not membrane-bound: inclusion bodies, ribosomes, numerous other proteins and the nucleoid with its genetic material.

5. Introduction The prokaryotic cell wall typically has a chemically and morphologically complex peptidoglycan layer. Most bacteria can be divided into Gram-positive and Gram-negative groups based on differences in the permeability of the two essential cell wall types and response to the Gram stain.

6. Introduction They have 70s ribosomes within the cytoplasm giving it a granular appearance. Have a Nucleoid made of single stranded DNA lying freely in the cytoplasm without a nuclear membrane. Have components like capsule and fimbriae and flagellum, located outside the cell wall. Some bacteria form resistant endospores to survive harsh environmental conditions in a dormant state.

7. Differences between prokaryotes and eukaryotes Eukaryotic Cell Prokaryotic Cell Nucleus present absent Number of chromosomes More than one One but not true chromosome True Membrane bound Nucleus Present Absent Examples Animals and Plants Bacteria and Archaea Plasma membrane with steroid Yes Usually no Cell wall Only in plant cells and fungi (chemically simpler) Usually chemically complex Lysosomes and peroxisomes Present Absent Microtubules Present Absent or rare Endoplasmic reticulum Present Absent Mitochondria Present Absent Cytoskeleton Present May be absent Ribosomes Larger smaller

8. Differences between prokaryotes and eukaryotes contd. Eukaryote Prokaryote Vesicles and vacoules Present Present Golgi apparatus Present Absent Chloroplasts Present (in plants) Absent Cell Type Usually multicellular Usually unicellular(some cyanobacteria may be multicellular ) Cell size 10-100um 1-10um Genetic Recombination Meiosis and fusion of gametes Partial, undirectional transfers of DNA Permeability of Nuclear Membrane Selective No nuclear membrane

9. Bacterial Taxonomy Example : Kingdom – Prokaryote Division – Gracilicutes Class – Scrotobacteria Order – Eubacteriales Family – Enterobactericeae Genus – Proteus Specie – vulgaris, mirabilis

10. Size, shape, & arrangement of bacteria Bacteria are small, and vary in size (500 – 800nm) At the lower end , some bacteria ( rickettsias , chlamydia, and mycoplasmas) overlap with the largest viruses (the poxviruses), and at the upper end, some rod-shaped bacteria have a length equal to the diameter of some eukaryotic cells.

11. Size, shape, & arrangement of bacteria Bacteria vary in shape and arrangement: Cocci – roughly spherical cells. May occur: Singly I n pairs ( diplococci , divide and remain together to form pairs e.g. Neisseria spp , Streptococcus pneumoniae ) In chains (when cells adhere after repeated divisions in one plane e.g. Streptococcus , Enterococcus ) In grapelike clusters (when cells divide in random planes e.g. Staphylococci ).

12. Size, shape, & arrangement of bacteria Bacilli or Rods: are rod shaped. Differ in their length-to-width ratio e.g. - Straight (majority of them are like this; e.g. E.coli ) C occobacilli are so short and wide that they resemble cocci e.g. Haemophilus spp., Acinetobacter spp. Some rod-shaped bacteria are curved to form distinguished commas or incomplete spirals e.g. Vibrio spp ., Campylobacter spp.

13. Size, shape, & arrangement of bacteria - S lender: Mycobacterium tuberculosis - Robust: Lactobacilli , Bacillus spps - With split ends: Bifidobacteria Branching: Nocardiae , Actinomycetes With flat ends: Bacillus anthracis S pindle-shaped: Fusobacteria C lub-shaped: Corynebacteria P leomorphic: Haemophili

14. Size, shape, & arrangement of bacteria cont’d Spirilla and Spirochetes: long rods twisted into spirals or helices e.g. Treponems , Borreliae and Leptospirae species. Thick : Spirillium Uneven : borrelia Delicate , regular: Treponema Slender with bent ends: Leptospira

15. Size, shape, & arrangement of bacteria cont’d Mycelium (filamentous): long, multinucleate filaments or hyphae e.g. Actinomycetes . Pleomorphic : bacteria that vary in shape and lack a single, characteristic form e.g. Corynebacterium .

16. Size of bacteria Unit for measurement : Micron or micrometer ( μm ): An average bacterium is about 1μm wide (10-3mm) Size: Varies with kinds of bacteria, and also related to their age and external environment. Cocci : sphere, 1μm Bacilli: rods , 0.5- 1 μm in width 1- 3 μm in length Spiral bacteria: 1~3 μm in length and 0.3-0.6 μm in width

17. 17 Rods (bacilli) cocci spirochetes http://www.scientificpsychic.com/health/hygiene.html Spirillum http://www.daviddarling.info/images/spirillum.jpg Filamentous www.theguardians.com/Microbiology/begg1_bg.jpg Square www.boingboing.net/bacteria.jpg

18. Basic structures Cell wall Cell membrane Cytoplasm Nuclear material Special structures Capsule Spore Flagella Pili

19. 19 Overview of prokaryotic cell.

20. Structure of Bacteria Particular structure c apsule flagella pili spore Essential structures cell wall cell membrane cytoplasm nuclear material

21. Cell wall Situation: outmost portion. 15-30nm in thickness, 10%-25% of dry weight.

22. Bacterial Structure: Cell Wall Comprises the layers between the capsule and the plasma membrane viz : A. Outer membrane is a lipid bilayer found only in GNB. It contains lipopolysaccharide ( LPS ) AKA endotoxin found only in GNB and Listeria monocytogenes LPS is synthesized on the plasma membrane then transported to its final exterior position LPS is composed of 3 parts – lipid A (responsible for its endotoxic activity), polysaccharide (O antigen which confers antigenic specificity) and the core (similar in all GNB) LPS are cell bound, heat stable toxins released from lysed cells

23. Cell Wall contd B. Peptidoglycan layer: Also called mucopeptide or glycopeptide or murein layer Peptidoglycan ( murein ), is an enormous mesh-like polymer composed of many identical subunits of alternating linkages of molecules of the two sugar derivatives, N-acetyl glucosamine (NAG) and N- acetylmuramic acid (NAM) in repeating dissacharides and several different amino acids in a side chain extending from each dissacharide . GPC have a thick continuous peptidoglycan(PG) layer while GNB have a thin discontinuous peptidoglycan layer Gram staining reaction depends on the difference in thickness of the peptidoglycan layer. Cell walls are absent in Mycoplasmas and L forms of bacteria(protoplasts and spheroplasts)

24. Cell wall : Common peptidoglycan layer A backbone of N-acetyl glucosamine and N- acetylmuramic acid : Both discovered in Gram positive and Gram negative bacteria. A set of identical tetrapeptide side chain attached to N-acetyl- muramic acid: different components and binding modes in Gram positive and Gram negative bacteria. A set of identical peptide cross bridges : only in Gram positive bacteria

25. Functions of Cell Wall Maintaining the cell's characteristic shape- the rigid wall compensates for the flexibility of the phospholipid membrane Countering the effects of osmotic pressure Providing attachment sites for bacteriophages Providing a rigid platform for surface appendages- flagella , fimbriae , and pili all emanate from the wall and extend beyond it Play an essential role in cell division Are the sites of major antigenic determinants of the cell surface 。 Target of some antibiotics which inhibit the synthesis of the peptidoglycan layer such as the β -lactams e.g. Penicillins , cephalosporins , monobactams , carbapenems. Site of resistance of antibiotics

26. Cell Wall Exceptions: Wall-less forms of Bacteria . When bacteria are treated with 1) enzymes that are lytic for the cell wall e.g. lysozyme or 2) antibiotics that interfere with biosynthesis of peptidoglycan, wall-less bacteria are often produced. Usually these treatments generate non-viable organisms. Wall-less bacteria that can not replicate are referred to as spheroplasts (when an outer membrane is present) or protoplasts (if an outer membrane is not present). Occasionally wall-less bacteria that can replicate are generated by these treatments (L forms).

27. Cell Wall Exceptions Mycobacterium and relatives Wall contains lots of waxy mycolic acids Attached covalently to Peptidoglygan

28. Special components of Gram positive cell wall : Teichoic acid Classfication : According to bonding site on the bacteria, teichoic acid can be classified into two types: Membrane teichoic acid, also called lipoteichoic acid (LTA) Wall teichoic acid SPA / M POTEIN

29. Functions of Teichoic acid Teichoic acid is made up of glycerol polymers that are bonded covalently to peptidoglycan and lipids. Its functions are as follows: It serves as a cell surface antigen, helping to identify and type a bacterium It is involved in the adherence of bacteria to host cell, relating to pathogenicity It promotes inflammatory response

30. Special components of Gram negative cell wall

31. Functions of outer membrane Composition: LPS, lipid bilayer, lipoprotein Adsorption and excretion Barrier function Pathogenicity Antigenicity Receptor for: F pilus , phage, bacteriocin

32. Special components of Gram negative cell wall Periplasmic space This cellular compartment is found only in those bacteria that have both an outer membrane and a plasma membrane or inner membrane in this case (e.g. Gram negative bacteria). In the space are enzymes and other proteins that help to digest and transport nutrients into the cell, substrate hydrolysis, electron transport and alteration of toxic substances

33. Bacterial Structure: Cell/Cytoplasmic/ Plasma Membrane Made of phospholipid and proteins in a ratio of about 3:1 However proteins make up 70% of the membrane mass GPB also have teichoic, lipoteichoic and teichuronic acids on their plasma membrane

34. Cell membrane Site of biosynthesis of DNA, cell wall polymers and membrane lipids. Selective permeability and transport (active and passive) of solutes into cells Electron transport and oxidative phosphorylation Chemotaxis - it bears specific receptors for chemo-attractants and repellents Biosynthetic functions – enzymes necessary for lipid and prostaglandin synthesis, enzymes and carrier proteins for cell wall synthesis and proteins required for DNA replication are located here

35. Mesosomes Mesosomes are specialized structures formed by convoluted invaginations of cytoplasmic membrane, seen in both gram-positive and gram-negative bacteria.

36. Mesosomes They are often found next to septa ( septal mesosome ) or cross-walls (lateral mesosomes ) in dividing bacteria and sometimes seen attached to the bacterial chromosome. Thus they may be involved in cell replication and distribution to daughter cells.

37. Cytoplasm Composed largely of water, together with proteins, nucleic acid, lipids and small amount of sugars and salts Ribosomes : numerous, 15-20nm in diameter with 70S ( made of 50s and 30s subunits) ; distributed throughout the cytoplasm; sensitive to streptomycin and erythromycin. - S ite of protein synthesis Plasmids : extrachromosomal genetic elements Inclusions : sources of stored energy, e.g. volutin

38. Bacterial Structure:Cytoplasm The genetic information of bacterial cell is mostly contained in a single, long molecule of double-stranded deoxyribonucleic acid (DNA). The cell solves the problem of packaging this enormous macromolecule by condensing and looping it into a supercoloid state. The bacterial nucleoid lies within the cytoplasm. This means that as DNA-dependent RNA polymerase makes RNA, ribosomes may attach and initiate protein synthesis on the still attached (nascent) messenger RNA . Synthesis of mRNA and protein are therefore seen to be directly coupled in bacteria .

39. Plasmid Plasmids are small ， circular / linear ， extrachromosomal ， double -stranded DNA molecules 。 They are capable of self-replication and contain genes that confer some properties ， such as antibiotic resistance ， virulence factors 。 Plasmids are not essential for cellular survival. Inclusions of Bacteria Inclusions are aggregates of various compounds that are normally involved in storing energy reserves or building blocks for the cell. Inclusions accumilate when a cell is grown in the presence of excess nutrients and they are often observed under laboratory conditions. granulose

40. Nucleus Lacking nuclear membrane, absence of nucleoli, hence known as nucleic material or nucleoid, one to several per bacterium.

41. Capsules and slime layers These are structures surrounding the outside of the cell envelope. They usually consist of polysaccharide; however, in certain bacilli they are composed of a polypeptide (polyglutamic acid). They are not essential to cell viability and some strains within a species will produce a capsule, whilst others do not. Capsules are often lost during in vitro culture. Attachment Protection from phagocytic engulfment. Resistance to drying. Depot for waste products. Reservoir for certain nutrients. protection

42. Flagella Monotrichate /Amphitrichate/ Lophotrichate/ Peritrichate Identification of Bacteria Pathogenesis Motility of bacteria Some bacterial species are mobile and possess locomotory organelles - flagella. Flagella consist of a number of proteins including flagellin The diameter of a flagellum is thin, 20 nm, and long with some having a length 10 times the diameter of cell. Due to their small diameter, flagella cannot be seen in the light microscope unless a special stain is applied. Bacteria can have one or more flagella arranged in clumps or spread all over the cell.

43. Flagella Bacterial species often differ distinctly in their patterns of flagella distribution and these patterns are useful in identifying bacteria: Monotrichous bacteria – have one flagellum. Called polar flagellum if located at an end e.g. Pseudomonas.

44. Flagella Amphitrichous bacteria – have a single flagellum at each pole e.g. Lophotrichous bacteria – have a cluster of flagella at one or both ends e.g. Spirillum . Peritrichous bacteria – have flagella spread fairly evenly over the entire surface of the bacteria e.g. Proteus vulgaris .

45. Pili Pili are hair-like projections of the cell . They are known to be receptors for certain bacterial viruses . Chemical nature is pilin Classification and Function Common pili or fimbriae : fine, rigid numerous, related to bacterial adhesion Sex pili : longer and coarser, only 1-4, related to bacterial conjugation

46. Endospores (spores) Dormant cells Resistant to adverse conditions - H eat - Chemicals - Desiccants - organic solvents Produced when starved Contain calcium dipicolinate DPA, D ipicolinic acid Bacillus and Clostridium Identification of Bacteria Pathogenesis Resistance

47. Staining Techniques Gram stain - Hans Christian Gram Ziehl-Neelsen stain Fluorescent stains – Auramine -Phenol stain for AFB Capsule stains – negative staining using congo red, india ink or nigrosin . In addition, positive staining with crystal violet or methylene blue will increase the contrast and aid better visualization Flagellar stains e.g. Leifson’s stain Spore stains – Malachite green and Safranin / Carbol Fuschin

48. Gram stain Primary stain - Crystal violet Mordant – Iodine Decolorizer – 70% Alcohol or Acetone Secondary stain – Neutral red, Safranin or dilute Carbol fuschin

49. Gram + Gram - Cell wall Cell (inner) membrane Outer membrane Ribosomes Granule Cell wall Nucleoid Cell membrane Capsule Flagellum Pili Gram, C. 1884. Ueber die isolirte Farbung der Schizomyceten in Schnitt Ä und Trockenpraparaten. Fortschritte der Medicin , Vol. 2, pages 185-189. 1884: Christian Gram : First publication for the Gram stain method)

50. Gram stain 53 Gram stain invented by Hans Christian Gram Divides Eubacteria into two main groups based on stain. Correlates with two types of cell wall architecture.

51. Division of the Eubacteria: Gram Negative and Gram Positive Stain is valuable in identification. Gram positives stain purple; Gram negatives stain pink. Architecture: Gram positives have a thick peptidoglycan layer in the cell wall; Gram negatives have a thin peptidoglycan layer and an outer membrane. 54

52. 55 http://www.conceptdraw.com/sampletour/medical/GramNegativeEnvelope.gif http://www.conceptdraw.com/sampletour/medical/GramPositiveEnvelope.gif Gram Negative Gram Positive

53. Differences between gram negative and Gram positive cell wall Gram positive bacteria Gram negative bacteria Have teichoic and lipoteichoic acids Do not Have no lipopolysaccharide (LPS) except Listeria monocytogenes Have lipopolysaccharide Have no periplasmic space Have periplasmic space Have thick continuous peptidoglycan layer Have thin discontinuous peptidoglycan layer Less resistant to antibiotics More resistant to antibiotics Have no porin channels Have porin channels Have only an inner cell membrane Have both inner and outer cell membrane Lipid and lipoprotein content is lower Have higher lipid and lipoprotein content Toxins produced are primarily exotoxins Toxins primarily endotoxins Appear blue after gram staining Appear red after Gram staining Highly resistant to drying and physical disruption Less resistant

54. ZN STAIN Primary stain – concentrated (strong) Carbol fuschin Decolorizer – 3% acid-alcohol , 1% acid- alcohol Secondary stain – Methylene blue or Malachite green

55. Bacterial Growth Bacterial growth may be seen as: Colonies on solid media Turbidity in liquid media Biofilm - thin film of microbial growth that is enclosed in an extracellular polymeric substance matrix, usually on an inert surface

56. Bacterial Growth curve

57. Bacterial Growth curve contd. Lag phase - no of cells remain constant, cells thought to be preparing for growth . S ynthesis of RNA , enzymes and other molecules occurs. Exponential (log) phase - rapid acceleration of growth rate to a maximum for the available conditions, there are more than enough nutrients to allow the cells to grow, increase in the cell count is detectable . Stationary phase - the number of cells remains constant as growth rate is constant; the number of cells being produced=number of cells dying . Here the growth rate slows down due to the reduction in available nutrients and the accumulation of metabolites . Decline(death) phase - reduction in growth rate and cell count due to exhaustion of nutrients and accumulation of metabolic by-products.

58. Bacterial metabolism This is the way in which a microbe obtains energy and nutrients it requires; to survive and reproduce. The processes can be anabolic (synthesis of compounds and the consumption of energy) or catabolic (break down of substrates to gain energy). Although some bacteria are able to obtain their resources for growth in many ways . The basic details of glycolysis, the tricarboxylic acid cycle, oxidative phophorylation , ATP biosynthesis and amino acid metabolism are constant. Human pathogenic bacteria are always chemosynthetic, organotrophic bacteria (or chemoorganotrophs ).

59. Bacterial metabolism Bacterial metabolism is affected by a number of factors which will be discussed subsequently. Thus bacteria may be classified based on some of these factors as follows:

60. Classification Based On Oxygen Use Obligate Aerobes: can only reproduce in the presence of oxygen. Have Catalase and Superoxide dismutase Able to break down highly reactive oxygen molecules such as hydrogen peroxide 2H 2 2 Catalase 2H 2 0 + 0 2 2 + 0 2 + 2H SOD H 2 2 + 0 2 Mycobacterium tuberculosis, Norcardia asteroides , Pseudomonas aruginosa , Bacillus anthracis

61. Obligate Anaerobes: die in the presence of oxygen. Their vital enzymes are inhibited by oxygen . Cannot survive in the presence of oxygen Bacteroides fragilis , Actinomyces spp., Prevotella Facultative Anaerobes: they oxidise nutrients by respiration and fermentation. They can grow in all conditions. E.g. aerobes that are capable of growth in the absence of oxygen using fermentation for energy production - Escherichia coli, Staphylococcus aureus Most medically important bacteria belong here

62. Micro- aerophiles These organisms require lower oxygen tension than is present in atmospheric air to survive They lack the catalase enzyme but have SOD Use fermentation for energy production Campylobacter spp , Helicobacter spp , Treponema spp , Leptospira spp , Borrelia spp., Streptococcus spp Aerotolerant anaerobes: they don’t utilise oxygen for growth but can survive in its presence. Capnophilic bacteria: they need higher amounts of CO2.

63. Classification Based on energy source and use of carbon Phototrophs – use light as their source of energy Chemotrophs – use chemical compounds as source of energy Autotrophs – utilise inorganic sources of carbon for energy e.g. ammonium and sulphide Heterotrophs – utilise organic carbon compounds (e.g. glucose) for energy. All medically important bacteria belong here

64. Classification Based on glucose metabolism Oxidative respiration (aerobic metabolism) Glycolysis, Kreb’s cycle(TCA), electron transport chain Fermentation (Anaerobic) Glucose - pyruvate – lactic acid, ethanol, propionic acid, butyric acid, acetone etc. These products are used to classify anaerobes

65. Classification Based on temperature requirements Temperature affects a variety of bacterial properties including growth rate and viability Bacteria vary in their optimal temperature ranges. Extremes of temperature can kill micro-organisms e.g. high temperatures, and rapid cooling (cold shock) Psychrophiles – grow best at 15-20 degrees celsius E.g. Listeria monocytogenes , Mycobacterium leprae

66. Classification Based On Temperature Mesophiles – grow best at 30-37˚C Most human pathogens and opportunists belong here Thermophiles – grow best at 50-60˚C e.g. Bacillus stearthermophilus

67. Based on pH requirement Most organisms are able to maintain a fairly narrow optimal pH range despite a wide range of external pH values. Based on this, organisms are grouped as: Neutralophiles – grow best at pH of 6.0-8.0 Most bacteria of medical importance belong here Acidophiles – grow best at pH of less than 5.5 Alkalophiles – grow best at pH of more than 8.5

68. Effects Of Osmotic Pressure Osmotic pressure arises when the concentration of solutes inside the cell differs from that outside Adaptive responses occur to minimal alterations viz : In hypotonic solutions- cellular swelling occurs In hypertonic solution – cell shrinkage (plasmolysis) The cell wall provides osmotic protection when the cell’s limit of adaptation is exceeded.

69. Effect of osmotic pressure on cells Hypotonic: water rushes in; PG prevents cell rupture. Hypertonic: water leaves cell, membrane pulls away from cell wall. 73 plasmolysis

70. Effect of Osmotic Pressure contd. However, when the cell wall of bacteria are treated with penicillin or lysozyme, they are affected thus: In GPB- complete loss of cell wall – Protoplast In GNB-PG layer lost but OM persists- Spheroplast These forms become osmotically sensitive and are no longer able to withstand osmotic pressure changes as before.

71. Microscope Light Microscope Electron Microscope Darkfield Microscope Phase Contrast Microscope Fluorescence Microscope Cofocal Microscope） Methods Staining Methods Simple staining; Differential staining (Gram stain, Acid-fast stain ) Special staining( Negative stain, Spore stain, Flagella stain)

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