Bacterial Structure

Aims and Learning outcomes: To describe the structure and function of bacterial flagellae, pili and fimbriae, capsules, ribosomes and storage granules. To describe the formation of endospores and their subsequent germination.

Flagella
Long thin appendages that arise at the level of the cytoplasmic membrane and extend through the wall into the surrounding medium. Enable bacteria to be mobile. Very thin, 20nm, not visible by light microscope unless stained.

Flagella
Seen mostly in rod shaped Gram negative bacteria and in Gram positive rods. Vary in number and arrangement on the cells. Polar or peritrichous. Characteristic in classification. Composed of flagellin protein, 30-40 Kda.

Flagella
Structure of flagella: not straight but helical, constant distance between 2 adjacent curves - wavelength, which is constant for a given organism. Composed of subunits of flagellin. Complex structure in Gram negative bacteria.

Flagella
Three main parts of the structure: the motor called the basal body, the hook (consisting of a single type of protein) at the base of the flagellum connecting the filament to the motor. The filament itself.

Flagella: basal body.

Basal body is anchored in the cytoplasmic membrane and cell wall. Small rod passing through a system of rings. In Gram negative bacteria outer ring in LPS, one in peptidoglycan and third in the cytoplasmic membrane. In Gram positive bacteria only the 2 inner rings present.

Flagella:Basal body
2 proteins called Mot are anchored either side of the S-M ring and drive the flagellar motor, causing a torque that rotates the filament. The Fli proteins act as a motor switch. Energy supplied by the passage of protons from outside the cell into the cytoplasm via the basal body mot complex. 1000 protons per single rotation.

Flagella: genetic control

In E.coli and S.typhimurium over 40 genes necessary for motility. Called fla, fli and flg. Several functions: structural proteins, exporting of flagellar components through the cell wall and biochemistry of synthesis. Flagella grow from the tip, flagellin moves up the hollow core.

Flagella: motility.
Enables movement at rate of up to 60 cell lengths per second. (faster than a cheetah) Movement different in polar and peritrichous flagella. Peritrichous in straight line, clockwise, tumbling, counterclockwise smooth swimming. Polar, more rapid spinning.

Flagella antigens and phase variation.

Salmonella spp. typed by their O somatic and H flagellar antigens. >2000 serotypes. Have the ability to differentially express the protein antigen as either phase 1 or phase 2 - phase variation.

Motility and chemotaxis.

Normal movement is randomly composed of runs and tumbles Chemotaxis -Response to different chemical gradients. Compare the chemical state of environment to that sensed seconds before and if attracted move up the gradient. Movement away if the chemical is repellant.

Pili and fimbriae.

Fine hair like filaments on the surface of many Gram negative bacteria. Not involved in motility. Fimbriae composed of 20 Kda protein. Pili generally longer. Both involved in attachment of bacteria to host cells. Bind to lectins on cell surfaces,e.g mannose. Gives tissue specificity.

Pili and fimbriae.

Bacteria within a human host are constantly losing and reforming fimbriae because fragile structures. Aids evasion of the immune system as bacteria, e.g. N. gonorrhoea change the antigenic structure of their pili/fimbriae.

Sex Pili and conjugation.

Used by conjugative plasmids to transfer copies of itself to a new host. F+ and F Sometimes other genetic material is mobilised during conjugation. After conjugation the previously negative cell has a copy of the plasmid and is able to pass it on to another cell. Efficient manner for transfer of antimicrobial resistance.

Sex Pili and conjugation.

The F plasmid of E.coli can also mobilise the chromosome to be transferred through cell to cell contact. Episome -integrates into the chromosome and leads to transfer of large regions of the host genetic material and extensive genetic recombination. F+ and F- strains. Cells already containing a plasmid are poor recipients for the same or similar plasmid.

Capsules and Slime layers

Consist of polysaccharide or sometimes protein, general term glycocalyx. Capsule: thick viscous gel outside and attached to cell wall. Slime: Capsule loosely attached to cell wall and easily washed off. Hydrophilic. Appear as halos around cells in a Gram stain

Capsules and Slime layers

Variable thickness e.g. Klebsiella spp and Strep pneumoniae up to 10m thick. Microcapsule of E.coli and Salmonella spp.too thin to be seen on light microscope, detected by antisera. Capsule not always expressed in vitro.

Composition of Capsules
Mainly acidic polysaccharide. The acidic groups being glucuronic acid or phosphate. Most are immunogenic and stimulate host antibody response. Exceptions are Group B meningococci capsule contains N-acetylneuraminic acid which is found on host cells. Hyaluronic acid of S.pyogenes capsule can lead to host cell damage because found on host cells.

Capsules and Slime layers:advantages to bacteria.

Protect cells from dessication and toxic material. Attachment to host cells. Soluble material released into solution blocks opsonising antibodies. Resist Complement alternative pathway. Resist phagocytosis by neutrophils.

Chromosome.
Single circular chromosome of double stranded DNA,called a nucleoid, not membrane bound, 300 to 400 m in length. Supercoiled like a rubber band. Genes arranged linearly along the chromosome. Size ranges from 600 to 9500 Kb. Composed of nucleotides, adenine, guanine, cytosine, thymine (uracil in RNA)

Protein Synthesis
Takes place on ribosomes, 70s (30s +50s) in the cytoplasm. 30s contains 16s RNA, 50s contains 23s RNA Up to 10,000 ribosomes per cell. Up to 35% of dry weight is RNA in growing cells. Bacterial mRNA is polycistronic ie. can be translated by several ribosomes simultaneously.

Protein Synthesis
Protein translation via tRNA, mRNA and 30s subunit initiation complex + 50s subunit Location of protein encoding genes determined by looking for Open reading frames. Start codon, codons and stop sequence.

Cytoplasm - carbon storage polymers

Amorphous gel containing enzymes and ions and a variety of granules, accumulation of food reserves (polysaccharides, lipids or polyphosphates). One of the most common inclusion bodies consists of poly--hydroxybutyric acid (PHB). Used as storage for carbon and energy storage polymers.

Cytoplasmic inclusions
Glycogen also used as storage product, starchlike polymer of glucose subunits. Smaller than PHB granules.
Plasmids and transposons. Transposons - jumping genes can insert into genome. Can also be transmitted with plasmids.

Endospores
Two genera of medical importance produce highly resistant endospores, which enable them to survive under adverse environmental conditions for long periods of time : Bacillus and Clostridia. Spherical or oval structures, dormant or resting phase. Survive in soil for up to 30 years e.g. B.anthracis, C.tetanus, C.perfringens.

Endospores
Resistant to drying, heat, pressure and many chemical disinfectants. Killed by heating to 120oC for 15-20 mins. Size, shape and location of spores within stationary phase of vegetative cells are helpful for identification. Appear as non-staining refractile areas within the cell

Endospores
Formation of spores stimulated by environmental conditions - lack of nutrients, change of temperature or redox potential. Several stages (see handout) Germination: converts back to vegetative cell relatively rapidly. 3 stages, activation, germination and outgrowth.

Endospores: Germination.
Activation: in vitro accomplished by heating at sub-lethal but elevated temperature. Germinate when placed in nutrients. Loss of resistance to heat and chemicals, loss of calcium dipicolinate and cortex components. Spore acid-soluble spore proteins degraded.

Endospores: outgrowth
Visible swelling as a result of water uptake and synthesis of new RNA, proteins and DNA. Cell emerges from broken spore and begins to divide.