Bacterial Culture & Growth

• Bacteria can be grown in liquid medium or on agar medium

Culturing Bacteria

• Nutrient broth is a rich media derived from yeast extract and casein
• LB (Luria Broth) is a type of nutrient broth used in practical classes
• Minimal media includes Na+, K+, Mg2+, Ca2+, Fe2+, NH4+, Cl-, phosphate buffer pH7, and glucose as a carbon source

Bacterial Cell Division

• Bacterial cells grow in size, replicate their chromosome, and divide into two identical daughter cells when sufficient nutrients are available
• E. coli can divide once every 20 minutes under optimal conditions; other species take longer

Bacterial Growth in Culture

• When placed in liquid growth medium, bacteria start to divide and grow
• Lag phase is the initial period when bacteria adapt to the new media
• Exponential (or log) phase is a period of rapid growth where bacteria divide at a fixed time interval called the doubling time
• Stationary phase occurs when cells stop dividing due to limited nutrients

Growing Bacteria on Plates

• Cells are streaked or spread on an agar plate, placed in an incubator, and divide
• The cells are immobile; a visible mass (colony) forms when ~10^7 progeny are reached from a single cell
• Colony members are clones, genetically identical due to derivation from a single genetic ancestor

The Bacterial Genome

• Bacteria typically possess a single, circular double-stranded DNA chromosome
• Bacteria have only one copy of each gene (haploid)
• There exhibits little intergenic space and no introns
• Functionally related genes are located in groups called operons, producing a single mRNA molecule
• Plasmids are not contained by all bacteria

Escherichia coli

• Escherichia coli (E. coli) serves as the best understood bacterial species and a model genetic organism
• Genetic principles were initially understood in E. coli before application to other organisms
• In 1997 the first complete DNA sequence of an E. coli genome was determined
• Genome is a circular, double-stranded DNA, 4,600,000 base pairs (4,600 kb)
• Genome codes for: 4288 protein-coding genes organized into 2584 operons, 7 ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA) genes
• E. coli is widely used by molecular biologists to create new recombinant DNA molecules called plasmids

Media Types and Genotype in E. coli

• Wild-type E. coli grows on minimal media (MM) with Na+, K+, Mg2+, Ca2+, Fe2+, NH4+, Cl-, phosphate buffer pH7, and glucose
• arg- E. coli can NOT grow on minimal media because it cannot make arginine; growth occurs only with arginine supplementation in the media
• Same principle is applicable for all 20 amino acids which E. coli can synthesize

Mutant Phenotypes

• Some mutant phenotypes require changes or supplements to minimal media
• lac+ E. coli grows on minimal media with lactose instead of glucose as the carbon (energy) source
• lac- E. coli will NOT grow on minimal media with lactose due to inability to utilize lactose as a carbon source

Genetic Exchange

• Genetic (DNA) exchange occurs between bacteria by:
  • Transformation: uptake of DNA fragments from the environment
  • Transduction: bacteriophage packages and transfers a DNA fragment from the bacterial genome to another
  • Conjugation: transfer of some plasmids between cells or transfer of bacterial genome when a conjugative plasmid integrates

Transformation

• Bacteria uptake DNA from their environment
• Bacteria can uptake linear, double-stranded DNA fragments from lysed/dead bacteria
• Linear DNA fragments cannot replicate independently and are targeted by cellular nucleases
• Similar uptake occurs with plasmid DNA, which then can replicate independently

Competency

• Very few bacteria in a natural population can uptake DNA via transformation
• Competency to uptake DNA depends on growth stage; varies
• In research, competency enhancement occurs through growing E. coli cells in rich liquid culture, harvesting during exponential growth, and treating with salts (ex CaCl2)

Transforming Plasmids

• Plasmid transformation into chemically competent E. coli cells occurs via incubation, heat shock (ex, 42°C for 30 sec), and recovery
• Transformed E. coli cells are spread on selective agar media plates
• selective agar media plates are Incubated at 37°C for 24 hours
• Colonies that grow on selective media plates are then observed

Transduction

• A bacterial virus (bacteriophage) packages a DNA fragment from the bacterial genome to transfer it to another cell

Bacteriophages

• Bacteriophage particles consist of an icosahedral head, a hollow protein sheath, and sometimes tail fibers
• The head contains a single chromosome of 5,000-100,000 base pairs
• Replication and gene expression require enzymes and host cell factors

Generalized Transduction

• Virus picks up a piece of the host genomic DNA and transports it to another cell

Viruses as Vectors

• Bacteriophages package DNA (or RNA in RNA viruses) of the appropriate length into mature virus particles automatically
• This knowledge has led to the use of viruses as vectors for recombinant DNA molecules facilitating research and gene therapy

Conjugation

• Conjugation is the transfer of some types of plasmids between bacterial cells, or transfer of the bacterial genome between bacterial cells
• The transfer occurs when a plasmid capable of conjugation is integrated into the bacterial genome

Conjugation Discovery

• In 1946 Lederberg and Tatum mixed two E. coli strains with different nutritional requirements; they recovered recombinants able to grow on minimal media
• This discovery suggested that mutant E. coli strains exchange DNA to give full wild-type bacteria

Cell Contact Required

• Microscopic studies confirm that physical union of bacteria is required during conjugation
• During conjugation, the donor cell transfers genetic information to a recipient cell
• Genetic information is transferred through a hollow tube called a conjugation pilus, conjugation tube, or sex pilus made of proteins

F (Fertility) Plasmid

• The F (fertility) plasmid of E. coli was the first plasmid discovered
• F plasmid carries genes enabling genetic exchange by conjugation
• F plasmid is 100 kb in size and has following features:
  • Approximately 30 transfer (tra) genes
  • An origin of replication (oriV)
  • An origin of transfer (oriT)
  • Three insertion sequences (IS)

F+ Cell

• The F+ cell has a conjugation pilus that attaches to the F- cell and retracts, drawing the cells closer to form a tube composed of pilin proteins
• Transfer of a single strand copy of the F plasmid occurs from the F+ cell to the F- cell
• It takes 2 minutes for the F plasmid to transfer into the F- cell
• Before transfer, a single-strand nick is produced at oriT in the F plasmid by the relaxosome (protein complex)
• Copy of a single DNA strand (T strand) transfers into the F- cell via conjugation pilus
• DNA replication ensures both daughter cells become intact, circular, double-stranded DNA molecules
• Each cell then has an F plasmid, being referred to as F+

Hfr Strains

• Approximately 1 in 100,000 E. coli cells have F plasmid incorporated into the genome
• E. coli cells which have a copy of the F plasmid in their genome are known as Hfr strains
• Hfr stands for high frequency of recombination
• F plasmid integration occurs only when with homologous recombination between IS sequences on F plasmid and bacterial chromosome

Integrated F Plasmid

• The bacterial chromosome can be mobilized by the integrated F plasmid
• An Hfr cell can conjugate with an F- cell and bacterial DNA flanking integrated F plasmid transfers across into F- or recipient cell
• Transfer of the entire E. coli chromosome would take 100 minutes
• Mating E. coli pair usually break apart before the whole chromosome is transferred (link is delicate) so only genes near the F plasmid insertion point are transferred
• Recipient (F-) cell remains F- due to broken 'mating'
• For the transferred DNA to survive in a recipient cell it must integrate via homologous recombination into the chromosome because it cannot replicated itself independently

R Plasmids

• R plasmids are naturally occurring plasmids found in a range of bacterial species
• R plasmids have tra genes and they conjugate between different bacterial species
• R plasmids carry genes that confer multiple antibiotic resistances
• R plasmids are approximately 100 kb in size

ColE1 Plasmids

• ColE1 plasmid is found in E. coli
• It is small (6 kb), doesn't carry tra gene cluster, so it does move by itself between different bacteria
• The origin of colicin E1 is often used in engineered plasmids used in molecular biology
• The ColE1 plasmid carries ColE1 gene that produces colicin E1 protein excreted from the cell and toxic to other bacteria (not the host)

Nobel Prize

• The Nobel Prize in Physiology or Medicine 1958 was awarded to Joshua Lederberg and George Wells Beadle and Edward Lawrie Tatum for discoveries concerning genetic recombination and the organization of the genetic material of bacteria.