Bacteria 1

Classification of bacteria

• determined using microscopic, macroscopic and biochemical characteristics

  • size

  • staining property

    • gram-positve

    • gram-negative

  • shape

    • cocci (round)

    • bacilli (rod)

    • spirilla (helical)

    • fusiform (pointed rod)

    • cluster

    • chains or pairs

  • respiration

    • aerobic

    • anaerobic

  • reproduction

    • sporing

    • non-sporing

  

• important subspecies groups (biotype, strain, group) are identified based on

  • immunological properties

    • how they react with the immune system

    • serogroups and serotypes

      • defined by the bacteria cell wall, flagellar and capsule antigens (test with specific antisera——blood serum containg antibody)

  • biochemical characteristics

    • example: certain strain of Staphylococcus aureus release a β-haemolysin (toxin that causes RBC to lyse)

  • antibiotic susceptibility

  • phage typing

    • identifies bacterial strains based on their susceptibility to specific bacteriophages

    • example: differentiate between isolates of Vibro cholerae and Salmonella enterica serovars

  • Direct genetic approach

    • using PCR and probes to detect organism-specific sentinel (disease-causing) DNA sequence

Structure of bacteria

Cell wall

General Characteristics

• peptidoglycan——a mixed polymer of hexose sugars and amino acid: the main component of cell wall

• can be digested by lysozyme

• cell wall is the major contributor to the ultimate shape of an organism

• synthesis of peptidoglycan is distrupted by beta-lactam and glycopeptides antibiotics

• all bacteria except mycoplasmas are surrounded by cell wall

• might hv an addtional capsule

  • high molecular weight of polysaccharides → slimy suface

  • ✔︎ protection against phagocytosis

  • important in deteremining virulence

Gram staining

• Gram-positive

  • thick layer (20-80nm) of peptidoglycan layer

  • external to cell membrane

  • contain other macromolecules

    • lipoteichoic acids (LTA)

  • ★Mycobacteria

    • diff chemical basis for cross-linking to lipoprotein layer

    • outer layer envelop contain various complex lipids (mycolic acid)

    • cell wall compenents hv a pronounced adjuvant activity——promote immunologic responsiveness (can be used to enhance vaccine)

• Gram-negative

  • thin peptidoglycan layer (5-10nm)

  • overlaid by an outer membrane

    • lipopolysaccharides(LPS)

      • O antigen (carbohydrate chain): antigenic property

      • endotoxin (lipid A component): toxic property

    • lipoprotein

    • porin

      • for entry of hydrophilic molecules

    • anchored to lipoprotein molecules

Flagella

• log helical filaments extending from the cell surface

• enable motility of bacteria

• ╳ utilising ATP

• built of flagellins (protein): strongly antigenic

  • H antigen: important targets of protective antibdy responses

Pili (fimbriae)

• more rigid than flagella

• sex pili

  • attachment to other bacteria

  • for transfering DNA material

• common pili

  • attachment to host cell

• help prevent phagocytosis

  • antigenic variation: antigen changes

  • involve recombination of genes coding for ‘constant’ and ‘variable’ regions of pili

  • avoid immune variation

Nutrition

• take up small molecules across cell wall

  • uptake and transport by cell membrane via

    • facilitated diffusion by carriers

    • active transport

  • e.g. amino acid, oligosaccharides, small peptides

• Gram-negative bacteria can take up larger molecules

  • preliminary digest in periplasmic space (rich in enzyme)

• some species require

  • only minimal nutrients

    • e.g. E. coli can grow with glucose and inorganic salt only

  • complex nutrients

    • e.g. Streptococci grow only in complex media with many organic compounds

General nutritional requirement

Respiration

• requirement of oxygen can be obligate or facultative

  • aerobic

    • final product: 38 ATP+CO2+H2O

    • final electron acceptor: oxygen

  • anaerobic

    • final product: 34 ATP+NH3+H2S, etc (less ATP)

    • final electron receptor: other organic or inorganic molecule

    • less efficient but useful when absence of oxygen and other substrate are available

      • typically in host’s body

    

• mode respiration and ability of bacteria grow in the presence of O2 relates to their ability to enzymatically deal with potentially destructive intracellular reactive oxygen species

  • whether it has oxygen detoxifying enzymes

    • superoxide dismutase

    • calatase

    • perodidase

  • examples of reactive oxygen species

    • free radicals

    • anions with oxygen

    • superoxide

    

Growth and division

• depends large part on the availability of nutrients and genetical factor

  • Example:

    • E. coli double every 20min in lab vs 1-2 hours in a nutritionally depleted environment

    • Mycobacterium tuberculosis divide every 24 hr

• Bacterial growth curve

  • lag phase

    • period of adjustment when introduced to a new environment

    • sense the new environment→alter gene→synthesis of favorable protein for the specific environment to grow

  • log or exponential phase

    • population double in a constant rate

  • stationary phase

    • nutrients are depleted and toxic product accumulate

    • →equalise state: cell growth slows down and stops

  • death phase

    

Genomic DNA

• characteristics

  • all bacterial genomes are circular

  • DNA has no introns

  • ╳ nucleus → tightly coiled in to a nucleoid

  • genetic info can be extrachromosomal: encoded in plasmid (a small circular self-replicating DNA molecule)

• replication process

  • begins at a single site——OriC: origin of replication

  • multienzyme replication complex bind to OriC → inititate unwinding and separation of DNA strands

    • helicases (separate DNA strands)and topoisomerases (release DNA from supercoiled form, e.g. DNA gyrase) are used

  • DNA polymerase helps incorportation of DNTPs

• replication must be accurate

  • importance: DNA has info defining the properties and processes of cells

  • ✔︎ proofreading mechanism

    • base selection

    • 3’-5- exonuclease

    • mismatch repair

  • → reduce frequency of error ~ 1 per 10¹⁰

Gene expression

• majority of genes are transcribed into mRNA

  • e.g. up to 98% in E. coli

• some are transcribed to produce ribosomal RNA species

  • 5S, 16S, 23S

  • ✔︎ scaffold (platform) for assembling ribosomal subunit

• others are transcribed into transfer RNA

  • for decoding mRNA into functional proteins

Transcription

• frequency of trancription initiation is influenced by

  • exact DNA sequence of the promoter site

  • overall topology (supercoiling) of DNA

  • presence or absence of regulatory protein that bind adj to and may overlap the promoter site

• sigma factor

  • a component of RNA polymerase

  • important in promoter recognition

    • esp. in controlling exp of genes inv in spore formation in Gram +ve bacteria

  • several different factors allow sets of genes to be switched on

    • by altering level of expression of a particular sigma factor

• bacterial arrangement

  • monocistronic: one promoter and one terminator for one mRNA transcript

  • polycistronic

    • a single promoter and terminator flank multiple structural gene——operon

    • ≥ 1 protein is synthesed

    • importance: ensure protein subunits for particular enzyme complex or specific biological process are synthesised simultaneously and in the correct stoichiometry (ratio)

    • e.g.

      • uptake and metabolism of lactose are encoded by lac operon

      • cholera toxin from vibrio cholerae

      • pili of uropathogenic (pathogenic to urinary tract) E. coli → mediate colonisation

Regulation of gene expression

• importance: affect the ability of teh bacteria to adapt to changes in their environment

• highly regulate expression of many virulence determinants

  • to conserve metabolic energy

  • make sure virulence determinants are only produced when particular property is needed

• Example

  • enterobacterial pathogens (bacteria in guts): can adapt to environment like

    • low temp & low nutrients (water)

    • 37°C with rich supply of C and N but low O2 and free Fe (human guts)

  • adapt by switching on and off a range of metabolic and virulence-associated genes

• gene expression can be alter by changing the amount of mRNA transcription

  • alter the efficiency of binidng of RNA polymerase to promoter site

• activation: positive gene regulation——increase rate of transcription

  • binding of activator protein to operator site (where regulatory protein bind to)

  • binding of RNA polymerase to promoter

  • initiation of mRNA transcription

  • absence of activator protein → RNA polymerase fails to bind to promoter → no transcription

• repression: negative gene regulation——inhibit transcription

  • binding of repressor protein to operator site

  • inhibition of binding or activity of RNA polymerase

  • block mRNA transcription

  • absence of repressor protein → RNA polymerase can bind to promoter → allow inititation of mRNA transcription

• regulons: coordinated regulation of multiple genes

  • multiple genes are controlled by the same regulator protein (can be activator or repressor)

Translation

• ribosome bind to specific sequence of mRNA (Shine-Dalgarno sequences——upstream promoter sequence)

• begin to translate at AUG start codon

  • hybridise with specific complementary sequence of initiator of tRNA molecule

  • polypeptide chain elongation by condensation reaction of ribosome: couple the incoming amino acid to grow

  • termination at stop codon