Bacterial Cell Structure and 16S rRNA Sequencing

• Review of bacterial cell structure:

  • simple
  • prokaryote; no defined nucleus surrounded by membrane
  • DNA within nucleoid and plasmids

  A) Plasmid

  B) Nucleoid

  C) Ribosome

  D) Inclusions

  E) Capsule

  F) Cell Wall

  G) Plasma membrane

  H) S Layer

  I) Flagellum

  
• The nucleoid:

  • circular DNA
  • irregularly shaped due to large length
  • chromosome; main DNA essential for functioning
  • cell structure, nutrition, division etc
  • usually one per cell
  • highly organised
  • some have very big nucleoids e.g., Notsoc, some have very small e.g., Mycoplasma
• Plasmids:

  • small; closed, circular DNA
  • can be linear
  • vary in copy number (1-50)
  • can vary between species and strains
  • high number = metabolic burden but high chance will be passed to daughter cells
  • low number = no metabolic burden but unlikely to be passed to daughter cells
  • vary in length (2.3-1354kbp)
  • exist and replicate independently of chromosome
  • non-essential genes, usually conferring selective advantage e.g., drug resistance
• Plasmids are an important tool in molecular biology:

  • can be passed to other species; conjugation using pili
  • Insert plasmids containing certain genes into different bacteria to make them do things they wouldn't normally do
  • as long as they can be expressed, any gene can be inserted into a plasmid and therefore a bacterium
• Inclusions:

  • storage granules of organic or inorganic material for future use; stationary phase or in area of low nutrients
  • C, N and P most important
  • Can contain:
  • glycogen - C store; takes a while to breakdown, but low metabolic rate allows this
  • poly-beta-hydroxybutyrate (PHB) - lipid, C store; 100% biodegradable plastic, too expensive to mass produce, used in internal stitches
  • Phosphate - P store; takes a while to breakdown, but low metabolic rate allows this
  • Sulphur - anaerobic photosynthetic bacteria; hydrogen sulphide used as e- donor; elemental S used in stationary phase
• Cell wall:

  • either:
  • Gram-positive = 90% peptidoglycan
  • Gram-negative = 10% peptidoglycan
  • Gram staining:
  • bacterial smear on slide, flame to anneal
  • crystal violet added; binds to peptidoglycan
  • ethanol removes unbound crystal violet
  • Safranin dye added
  • Gram-positive = purple as crystal violet majority
  • Gram-negative = pink as safranin majority
  • look to the edge of smear for individual cells, not middle
• Gram-positive cell wall:

  • 90% peptidoglycan
  • Teichoic acid
  • carbohydrate similar to peptidoglycan (contains M and G)
  • can just be embedded in peptidoglycan
  • if embedded in plasma membrane = lipoteichoic acid
  • increase membrane fluidity
  • important receptors to phagocytes; allows macrophages to bind
• Gram-negative cell wall:

  • 10% peptidoglycan
  • thin layer of peptidoglycan; in periplasm = gap between inner and outer membrane
  • outer membrane complex:
  • porins = all G-neg. and some G-pos.
  • fully open to environment; small hydrophilic molecules and waste passage
  • targets for new antibiotics; starvation and toxic waste build up
  • no stand alone antibiotic yet
  • LPS = virulence
  • 3 main components:
  • lipid A = docked into membrane
  • core polysaccharide = consistent between species
  • O-antigen = highly variable polysaccharides; detectable by phagocytes; sugars can be changes making them unrecognisable
• Peptidoglycan structure:

  • Only found in bacteria
  • G = N-acetylglucosamine (NAG or GlcNAc)
  • M = N-acetylmuramic acid (NAM or ManNAc)
  • MG polymer chains linked by peptide bridges
  • targets for lysozyme; peptidoglycan structure lost; cell dies
• Bacterial Capsule: Extracellular polysaccharides (EPS)

  • capsule of polysaccharides
  • extent depends on species
  • protection from host defences e.g., phagocytosis
  • makes cells large and hard to absorb; ligands cannot be detected
  • protection from harsh environmental conditions e.g., dessication
  • overproduction e.g., slime = attachment to surfaces
• S-layers:

  • present in all archaea and some bacteria
  • monolayer of identical proteins or glycoproteins
  • functions - not entirely sure:
  • mutant with no s-layer = easier to phagocytose; mechanism unknown
  • anti-phagocytic
  • protrusions = extra layer helps attachment
• Flagella:

  • swimming and attaching to surfaces; 9+2 configuration (2 in centre, 9 outside); dynein motor
  • polar = at each end
  • monotrichous = one
  • amphitrichous = one at each end
  • lophotrichous = cluster at one or both ends
  • peritrichous = spread over entire surface
• Fimbriae and Pili:

  • Fimbriae
  • appendages; up to 1000 per cell
  • short, thin, hair-like, proteinaceous
  • recognition and attachment to surfaces
  • Pili
  • similar to fimbriae; longer and thicker
  • less numerous; 1-10 per cell
  • required for mating (sex pili; conjugation) and for attachment
  • Both can act as ligands allowing phagocytes to bind
  • bacteria and surfaces negative charged, hard to attach, need many mechanisms to bridge repulsion gap
• Natural Human Flora:

  • Majority of bacteria are attached; overcome flushing mechanisms
  • surface-associated mechanisms; biofilms
  • large surface area for attachment
  • will colonise different types of surface present e.g., soft tissue, enamel etc.
  • communities = mixed species
  • 90% of our cells are prokaryotic
  • 99% of DNA is prokaryotic
  • could not function without bacteria
• Why identify and classify bacteria?

  • sense of order to the diversity
  • enhance communication; ensure scientists are working and discussing the same organism
  • research
  • endemics/pandemics
  • provide means for accurate identification e.g., pathogens for diagnosis and treatment
  • molecular methods - 16S rRNA comparison
  • classic culture methods
• 16S rRNA sequence comparison (1):

  • S = sedimentation rate
  • each have their own
  • overall sedimentation of 70 due to conformation
  • 50S have 5S rRNA and 23S rRNA = highly conserved
  • 30S has 16rRNA = highly diverse

  
• 16S rRNA sequence comparison (2):

  • sequences from different species aligned and number of differences looked for
  • evolutionary distance = number of differences/total number of bases aligned
  • used to make phylogenetic trees
  • computer programs make complex trees
  • useful to identify unknown samples; will correspond to a species depending on its position in the tree
• Universal phylogenetic tree derived from 16S rRNA:

  • 16rRNA only in bacteria and archaea
  • eukaryotes have 18S; is referred to as 16S-like

  
• Archaea:

  • now further divided into archaea and Asgard archaea
  • genus names after Norse gods
  • very difficult to culture; only one to date
  • contain lots of enzymes found in eukaryotes and have similar pathways
  • eukaryota may have evolved from Asgard archaea