Bacteria

• Binary fission: Asexual means to produce offspring that are genetically identical to the parent
• process of binary fission:

  1. helicase separates the double helix at the ori site, forms a replication bubble made of 2 single DNA strands, replication takes place in both directions away from the origin
  2. 2 newly formed ori sites move to opposite poles of the cell, attaches to the cell surface membrane, cell elongates
  3. interlocking structure of 2 daughter dna molecules formed, topoisomerase cuts, separates and reseals the 2 dna molecules
  4. bacterium doubles in size, invagination of the cell surface membrane and deposition of new cell wall divides the parent cell into 2 daughter cells
• Horizontal gene transfer:

  • transformation
  • transduction
  • conjugation
• Transformation: uptake of naked, foreign DNA fragments from surrounding environment

  • competent cells contain surface membrane proteins that can bind to and transport DNA into cell
  • OR bacterial cells immersed in culture medium with high concentration of calcium chloride, followed by heat shock treatment
• why CaCl2 and heat shock needed:

  • bacterial cells permeable to chloride ions but not calcium ions
  • chloride uptake accompanied by influx of water, cells swell, heat shock induces formation of transient pores (for DNA fragment entry)
  • calcium ions bind to DNA to neutralise charge, allows cell to take up DNA
• Transformation process:

  1. foreign DNA incorporated into chromosomes through homologous recombination
  2. without homologous regions, foreign DNA degraded
  3. if different alleles are exchanged and expressed, cell has transformed, change in phenotype
• Transduction: phages randomly carry bacterial genes from one host cell to a recipient cell
• Generalised transduction:

  1. phage undergoes lytic cycle, phage enzymes hydrolyse host bacterial chromosome into small fragments of DNA that may contain genes
  2. small fragments of host bacterium degraded DNA may be randomly packaged within capsid head during assembly of virus
  3. defective phage released when cell lyses, infects another bacterium
  4. if homologous recombination takes place, foreign dna can replace
  5. recipient cell has new alleles integrated, will be a recombinant cell with diff genotype from previous host
• if any random portion of dna transferred -> generalised transduction
• specialised transduction:

  • carried out by temperate phages that undergo lysogenic cycle
  • bacterial DNA transferred is restricted to bacterial genes directly adjacent to the integrated prophage
• specialised transduction process:

  1. viral genome injected into host
  2. phage dna integrates into bacterial chromosome to form prophage
  3. upon induction, prophage exits bacterial chromosome, viral dna may be improperly excised, bacterial dna adjacent to prophage excised as well
  4. phage-host hybrid dna replicated, packaged into capsid heads
  5. recombinant phage infects other cell
  6. new alleles can be incorporated into bacterial genome via homologous recombination or integration of hybrid dna into genome of recipient cell
• conjugation: direct transfer of genetic material from once cell to another through a temporary link between the 2 cells

  • one way transfer, from F+ cell containing F plasmid to F- cell
• F plasmid contains a segment of DNA called F factor that carries the genes coding for sex pili
• conjugation procedure:

  1. sex pilis on F+ cell attaches to F- cell
  2. sex pilus retracts, pulls the 2 cells closer, mating bridge is formed between the 2 cells
  3. one strand of the double-stranded circular DNA nicked by nucleases, rolling circle dna replication takes place
  4. nicked strand transferred from F+ to F- cell via the mating bridge
  5. single strand F plasmid in F- cell recircularises, used as template to synthesise complementary strand for double-stranded F plasmid to form F+ cell
• rolling circle dna replication:

  1. one strand of double-stranded circular DNA nicked by nucleases
  2. DNA polymerase adds complementary nucleotides to free 3' -OH end of nicked strand by using un-nicked strand as template
  3. displacement of 5' end of nicked strand, nicked strand transferred to recipient bacterium via mating bridge
  4. newly synthesised strand displaces nicked strand
• benefits of conjugation:

  • gains new alleles, can be expressed to allow cell to survive in different environment
  • use of new resources
• operon: cluster of genes with related functions, contains common promoter and operator and produces a single polycistronic mRNA
• regulatory genes: codes for specific proteins that regulates the expression of other genes
• structural genes: gene that codes for a protein that forms part of structure or has an enzymatic function
• why operons are necessary in bacteria:

  • expression of structural genes regulated according to changes in environment
  • economical use of energy and resources, genes expressed only when necessary
  • allow for functionally related proteins to be synthesised as a unit, bacteria can respond rapidly and appropriately to changes in the environment
  • provides selective advantage
• Catabolic pathway: series of reactions that results in degradation of one or more specific cellular components
• Anabolic pathway: series of reactions resulting in synthesis of one or more specific cellular components
• Lac operon:

  • lacI: repressor protein produced in active form
  • lacZ: codes for B-galactosidase (hydrolyses lactose to glucose and galactose, converts lactose to allolactose)
  • lacY: codes for membrane transport protein permease that enables cells to take up lactose
  • lacA: codes for transacetylase

  
• inducer molecule for lac operon is allolactose (structural isomer of lactose)
• Lac operon is expressed in the presence of lactose and absence of glucose
• in the absence of glucose and lactose:

  1. lacI is transcribed to produce active lac repressor protein
  2. repressor protein binds to the lac operator at the protein’s DNA binding site
  3. Repressor blocks RNA polymerase and general transcription factors from binding to promoter

  however, repressor can dissociate sometimes due to weak interactions with operator, operon products can be produced
• in the presence of lactose:

  1. lactose enters the cell with the help of permease, converted to allolactose by few B-galactosidase molecules present
  2. allolactose binds to allosteric site of the repressor protein, inactivates it and alters the tertairy structure of the repressor, DNA binding site no longer complementary to operator and cannot bind to it
  3. RNA polymerase can access and bind to promoter
• positive regulation of lac operon: when glucose present, transcription of operon is repressed since cell would use up all the glucose first before metabolising lactose (energy is needed for expression of the operon), glucose used in preference to lactose as a respiratory substrate
• in the presence of lactose and glucose:

  1. lac operon has low affinity for RNA polymerase, even in presence of allolactose, lac repressor is not fully activated on its own
  2. low cAMP levels, CAP not activated, low binding affinity for CAP to promoter and thus low affinity of promoter region for RNA polymerase

  Cell has enough glucose to use for respiration, does not need to metabolise lactose to make glucose
• in the presence of lactose but absence of glucose:

  1. absence of glucose increase cAMP levels, cAMP binds to allosteric site of CAP to activate CAP
  2. CAP binds to CAP binding site within promoter, increases affinity of promoter region for RNA polymerase
  3. this increases rate of transcription of the operon
• Trp operon:

  • repressible operon (normally turned on until in the presence of tryptophan)
  • end product repression

  
• in the absence of tryptophan in the environment:

  1. tryptophan repressor synthesized in inactive form, RNA polymerase and general transcription factors free to bind to and transcribe the operon
• in the presence of tryptophan in the environment:

  1. tryptophan accumulates, binds to allosteric site of the repressor protein to activate it
  2. activated repressor binds to operator at the protein’s dna binding site, tryptophan serves as a co-repressor
  3. RNA polymerase and general transcription factors bind to the promoter, prevents transcription