CH 16 - Prokaryote Genetics

lecture 18 and 19

studies of bacteria were critical to the development of

the field of genetics

classical bacterial genetics (1940s1970s)

virtually all knowledge of gene structure, expression, and regulation came from studies of bacteria and bacteriophages

advent of recombinant DNA technology (1970s-1980s)

depend on understanding of bacterial genes, chromosomes and resitriction enzymes

all bacteria are prokaryotes, which

lack a define nuclear membrane and membrane-bound organelles

most bacteria have

a cell wall made of carbohydrate and peptide polymers that surrounds the cell membrane, single chromosome, rapid cell division

E. coli is the most studied and

best understood bacterial species

E. coli are

prokaryotic, lacks a defined nuclear membrane and membrane bound organelles

E. coli - lab strains are not

pathogenic, but other strains can cause variety or intestinal diseases

E. coli are phototrophic

can grow in minimal media and divides in one hour

E. coli genome is

tightly packed with genes; genome is one circular chromosome

genome of E. coli strain

• most encodes proteins

• no introns

• very little repetitive DNA

• small intergenic regions

individual E. coli strains contain

a subset of the E. coli pangeome

core genome -

about 1000 genes that are found in all strains

pan genome

core genome plus all genes that are found in some strains and not others

insertion sequences (IS elements) are like

eukaryotic transposable elements

Tn elements are

composite transposable elements

insertion sequences - have

inverted repeats at two ends

insertion sequences - carry

transposase gene, which imitate transposition by recognizing IRs

insertion sequences - can

move to other location in genomes, can disrupt gene function by insertion into their coding regions

insertion sequences - cause of

many spontaneous mutations

Tn elements are

compositie transposable elements

Tn elements have

two nearby transposable elements, which carry transposase gene

insertion sequences flank a gene conferring

resistance to antibiotics or toxic metals

plasmid are smaller

circles of ds DNA that carry genes beneficial to the host cell; can replicate independently of the bacterial chromosome

episomes

are plasmids that can integrate into the bacterial chromosome p

plasmids don’t carry genes

essential to the host; but may benefit host under certain stress conditions

plasmid genes beneficial to the hose

protect host against toxic chemical, metabolize environmental pollutants, pathogenic genes, resistance to antibodies

movement of antibiotic resistance gene to the plasmid was

facilitated by transposons

multiple antibiotic resistance genes can be transposed from

plasmid as a unit

bacteria must be grown and studies in

cultures

mutant variation in bacteria - altered colony morphology

large or small; shiny or dull; round or irregular

mutant variation in bacteria - resistance to bactericides

antibiotics, bacteriophages

mutant variation in bacteria - auxotrophs

unable to reproduce in minimal media

• defective in enzymes require to synthesize complex compounds

mutant variation in bacteria - defective using

• complex chemicals from environment (breaking down lactose into glucose and galactose)

• proteins essential for growth (conditional lethal mutations)

mutant alleles for bacteria

use a “-” superscript, or use a letter designating type of mutation

gene names for bacteria

three lower case letters, italicized

• leu genes for enzymes in leucine synthesis pathway

phenotype description for bacteria

capital letter, with no italics

• Leu- is a mutant for leucine synthesis that requires supplement of leucine for growth

rapid bacterial multiplication allows

detection of very rare genetic events

effectively haploid -

straightforward relationship between mutation and phenotypic variation

selection in bacterial genes

establish condition in which only the desired mutant will grow

• select for streptomycin resistance (Str-) ply plating on media containing streptomycin

genetic screen

examine each colony for a particular phenotype

most of the time, you do not know what the

consequences of a mutation, when looking at a plate of bacteria

lateral (or horizontal) gene transfer -

traits are introduced from unrelated individuals or from different species

vertical gene transfer occurs

in sexually reproducing organisms - traits are transferred from parent to offspring

three mechanisms for gene transfer in bacteria

transaction, transformation, conjugation

in all three mechanisms in gene transfer in bacteria - donor bacterium

provides the DNA that is transferred

in all three mechanisms in gene transfer in bacteria - recipient bacterium

receives the DNA, which can result in altered phenotype

genomic analysis has revealed widespread occurrence of

gene transfer mechanism in many bacterial species

gene transfer is an important mechanism for

rapid adaptation to environmental changes and to the development of pathogenic strains of bacteria

the gene encoding diphtheria toxin secreted by Corynebacterium diphtheria is carried by the genome of a lysogenic bacteriophage; the phage is acquired by

transduction, and then integrate its genome into host bacterial genome (horizontal gene transfer)

the F plasmid contains gene for

synthesizing connections between donor and recipient cells

conjugations

direct transfer of DNA from donor cell to connected recipient cell

donors for conjugation are

F+ (carry an special F plasmid)

recipients for conjugation are

F- (don’t carry an F plasmid)

conjugation process - F pilus binds to

F-cell wall

conjugation process - pilus retracts and

cells are drawn together

• then gene transfer

conjugation process - ater conjugation,

both cells are now F+ and can conjugate with other F- cells

F plasmid has three IS elements, which are

identical to IS elements found at various positions on the bacterial chromosome

cells with recombined F-plasmid called

Hfr

High frequency recombinant (Hfr) cells are formed when an

F plasmid integrates into the bacterial chromosome through recombination between IS elements

20-30 different Hfr strains can be generated that differ in

the location and orientation of the integrated F plasmids

integrated F plasmid replicates

with chromosomes during cell division

Hfr strains retain all

F plasmid functions and can be a donor for conjugation with an F- strain

transfer of DNA starts in the

F plasmid at the origin of transfer

chromosomal genes located to F plasmid sequences are

transferred to the recipient

transferre chromosomal DNA recombines into

homologous DNA in recipient

usually conjugation terminates before

entire chromosomes transfer

formation of F’plasmids

by excision from an Hfr chromosome

and F plasmid that can integrate into the bacterial genome is

an episomes

rare event - and integrated F plasmid comes out

of a Hfr chromosome

• and a few adjacent bacterial chromosomal DNA will be removed together with it

rare event - the newly formed plasmid carries most of the genes of the F plasmid plus some bacterial DNA,

known as an F’ plasmid or F’ episome

rare event - F’ plasmid can replicate

independently in bacterial cells

F’ plasmid can be transferred to

F- cells by conjugation

when an F’ plasmid transfers DNA to another bacterium, it can create

partial diploids (at certain gene loci)

merodiploids -

partial diploids in which two copies of some bacterial genes are available in the recipient cell (one copy on the F’ plasmid and the other copy in the bacterial chromosome)

• can be used for complementation tests

if the merodiploids shown can without tryptophan supplementation,

the two trp- mutations (x and y) are in two separate genes because tryptophan synthesis is a multi-step process

to select for Trp+ transfers,

plate on minimal media with histidine and no tryptophan

to select for His+ transfers,

plate on minimal media with tryptophan and no histidine

to screen for His+ Trp+ co-transfer,

test Trp+ individual and His+ individual bacteria for growth on minimal media with neither tryptophan nor histidine