Business

• Chapter 7: Problems 1-20,

– Master Solutions: Problems 1 and 2,

– Master Concepts in action: all,

• Questions about anything?

Bacteria Predominate

• 10,000+ “Species”,

– Mycoplasma genetalium • 200 nm

– Thiomargarista namibiensis• 750 m

– soil, water, air, symbionts,– have adapted to aquatic and

terrestrial extremes,

• 100 grams/person,

– 1014 bacteria.

• Metabolism;– Phototrophs,– Chemotrophs,

• Biochemistry;– ‘fix’ or synthesize a huge range of molecules,– break down almost anything,– adapt to just about anything.

• Molecular Biology;– Clone,– Gene therapy,– Eugenics,– Biotechnology,– Etc.

Bacteria Do Almost Everything

Bacterial Chromosome

...a circular molecule of double helical DNA,

– 4 - 5 Mb long in most species studied,

– 1.6 mm long if broken and stretched out.

• Inside the cell, the circular chromosome is condensed by super coiling and looping into a densely packed body termed the nucleoid.

Extra Chromosomal DNA

• Plasmids: circular double stranded DNA molecule that replicates independently,

– containing one or more (non-essential) genes, smaller than the bacterial chromosome,

– may carries genes for pathogenicity,

– may carry genes for adaptation to the environment, including drug resistance genes,

– 1000’s of base pairs long.

Bacterial Model Organism Escherichia coli = E. coli

• Enteric bacteria: inhabits intestinal tracts,– generally non-pathogenic,– grows in liquid,– grows in air,

• E. coli has all the enzymes it needs for amino-acid and nucleotide biosynthesis,– can grow on minimal media (carbon source and

inorganic salts),

• Divides about every hour on minimal media,– up to 24 generations a day,

Growth Equals Cell Division

DNA Replication

Binary Fission

Bacterial mitosis. Why don’t bacteria do meiosis?

The (Awesome) Power of Bacterial Genetics

... is the potential for studying rare events.

Liquid Cultures,

• 109cells/microliter,

Colonies on Agar,• 107+ cells/colony

Counting Bacteria

(Serial) Dilution is the Solution

10-3 10-510-4

Model Model Organism

• Ease of cultivation,Ease of cultivation,

• Rapid Reproduction,Rapid Reproduction,

• Small size,Small size,

• Fecund (large brood size),Fecund (large brood size),

• Mutants are available, stable and easy to identify?Mutants are available, stable and easy to identify?

• Literature?Literature?

• PubMed Listings: Eubacteria: 612,471, Archaebacteria: 9,420PubMed Listings: Eubacteria: 612,471, Archaebacteria: 9,420

Bacteria Phenotypes

• colony morphology,

– large, small, shiny, dull, round or irregular,

– resistance to bactericidal agents,

• cells unable to synthesize certain raw materials from a

minimal media,

• cells unable to break down complex molecules,

• essential genes, usually studied as conditional mutants.

Prototroph…a cell that is capable of growing on a defined, minimal

media (all essentials salts, carbon source),

– can synthesize all essential organic compounds,

– usually considered the ‘wild-type’ strain.

Auxotrophs…a cell that requires a substance for growth that can

be produced by a wild-type cell,

his- ...can’t synthesize histidine (his+ = wt) leu- ...can’t synthesize leucine (leu+ = wt)

arg- ...can’t synthesize arginine (his+ = wt)bio- ...can’t synthesize biotin (bio+ = wt)

Bacterial Nomenclature I

• genes not specified are considered wild-type,

Strain A: met- bio-

x

Strain B: thr- leu- thi-

thr+ leu+ thi+

met+ bio+

Complete Media…

Contains all essential nutrients, plus a carbon source, and supplemented with specific complex molecules,

- bacteria uptake molecules that it can not synthesize.

Minimal Media…

Contains all essential nutrients (usually as salts), plus a carbon source,

- bacteria synthesizes the

rest.

1 in 107

awesome

Strain A: met- bio- tri+ leu+ thi+

x

Strain B: met+ bio+ thr- leu- thi-

Conjugation

Strain C: met+ bio+ thr+ leu+ thi+

exconjugate

Conjugation

...temporary fusion of two single-celled organisms for the transfer of genetic material,

…the transfer of genetic material is unidirectional.

F+ Cells(F for Fertility)

… F+ cells donate genetic material.

… F- cells receive genetic material,

…there is no reciprocal transfer.

F- Cells(F for Fertility)

Contact is Required For Mating

F Pilus

…a filament-like projection from the surface of a bacterium.

F+

F-

F Factor

…a plasmid whose presence confers F+, or donor ability.

F Pilus Attaches to F- Cell

Cells are Drawn Together

Passageway Forms

DNA is Nicked, One Strand is Transferred, Both Strands Replicate

Single Strand Moves Across Pilus

The single stranded plasmids synthesize complementary strands in their respective cells .

Cell Separation

Both cells are now F+

F Factor Replicates During Binary Fission

Properties of the F Factor

• Can replicate its own DNA,

• Carries genes required for the synthesis of pili,

• F+ and F- cells can conjugate,– the F factor is copied to the F- cell, resulting in two F+ cells,

• F+ cells do not conjugate with F+ cells,

• F Factor sometimes integrates into the bacterial chromosome.

Hfr Cells

F factor

Bacterial Chromosome

Inserted F plasmid

...F factor integration site,

...host (bacteria chromosome) integration site.

...the bacterial chromosome contains many integration sites,

...thus, the F factor inserts in different regions of the bacterial chromosome.

High Frequency of Recombination(Hfr)

...bacteria exhibiting a high frequency of recombination,

– an alteration of DNA sequence such that the genotype of subsequent individuals differs from the parent,

…HFR cells; strains with a chromosome integrated F factor that is able to mobilize and transfer part of the chromosome to the F- cell.

F Pilus Attaches to F- Cell

Hfr DNA is Cut

F factor and Chromosomal DNA are Transferred

Recombination Requires Crossing over

Double Crossover

DNA not Incorporated into Chromosome are Digested

Incomplete Transfer of DNA

• Interrupted Mating: a break in the pilus during conjugation stops the transfer of DNA,

• Transfer occurs at a constant rate,– provides a means to map bacterial genes.

Hfr to F- Cells

• Hfr transfer begins in the middle of the F factor,

• Only 1/10,000 cells transfer the complete chromosome, including the remainder of the F factor,

• Thus, most recipient cells remain F-.

F’Cells

• an F factor from an Hfr cell excises out of the bacterial genome and returns to plasmid form,

• often carries one or more bacterial genes along,

• F’cells behave like an F+ cells,

– merizygote: partially diploid for genes copied on the F’plasmid,

• F’plasmids can be easily constructed using molecular biology techniques (i.e.vectors).

Mutation in Bacteria

…the ultimate source of variation in bacteria is spontaneous mutation,

– generally errors in DNA replication,

…mutations occur in specific genes at a rate of 1 in 106 to 1 in 107 cells,

…adaptive mutations are quickly replicated and adaptive colonies predominate.

Spontaneous Mutations

• DNA replication in E. coli occurs with an error every ~ 109 bases.

• The E. coli genome is 4.6 x 106 bases.

An error occurs once per ~ 2000 replications.

• If a single colony has 107 bacteria,

5,000 cells carry a mutation,

or, one mutation every ~ 1,000 bases (across a colony),

or, a mutation in about every gene.

Selective Media

• wild-type bacteria grow on minimal media,

• media supplemented with selected compounds supports growth of mutant strains,

– minimal media + leucine supports leu- cells,– minimal media + leucine + arginine supports leu- arg-

– etc.

• Selective Media: a media in which only the desired strain will grow.

Selection

...the process that establishes conditions in which only the desired mutant will grow.

Problem

Strain MatingType

Chromosome Genotype F factor Genotype

Strain 1 F' wild-type thr+ ; thi+

Strain 2 F' arg- thr+ ; thi+

Strain 3 F- thr- ; thi- ; met- -

You want to create a culture of cells that are only methionine auxitrophs?

The Wrong Answer

Strain MatingType

Chromosome Genotype F factor Genotype

Strain 1 F' wild-type thr+ ; thi+

Strain 2 F' arg- thr+ ; thi+

Strain 3 F- thr- ; thi- ; met- -

Strain 1 x Strain 3 (complete media)

How do you get rid of Strain 1?

You have a recombinant that is met- only.

Correct AnswerStrain Mating

TypeChromosome Genotype F factor Genotype

Strain 1 F' wild-type thr+ ; thi+

Strain 2 F' arg- thr+ ; thi+

Strain 3 F- thr- ; thi- ; met- -

Strain 2 x Strain 3 (complete media)

Grow on Minimal Media Plus Methionine

Strain 2 dies because there is no arginine.

Strain 3 dies because there is no threonine or thimine.

The new exconjugate lives.

F factor Review

Now F+ Now F+