Microbial genetics and reproduction

Microbial genetics and Microbial genetics and reproductionreproduction

By By Dr. C. Dr. C. RexachRexachMicrobiologyMicrobiology

Mt San Antonio CollegeMt San Antonio College

Definitions• Chromosome• DNA• Gene• RNA• Genotype• Phenotype

DNA structure• DNA composed of subunits =

nucleotides– Sugar, phosphate, nitrogen base (A, G,

C,T)• Double stranded• Anti-parallel structure

3’

5’

5’

3’

DNA structure

OC1

23

4

5

Base pairing rulesA=TC=G

DNA replication

• Occurs just before cell is going to divide = reproduction

• Semi-conservative process resulting in production of two identical daughter molecules

• Enzyme mediated

Steps of DNA replication• DNA unwinds and separates one segment

at a time, forming a replication fork• Free nucleotides in the cytoplasm matched

by DNA polymerase to exposed base pairs following base paring rules

• Nucleotides are added in a 5’3’ direction = bidirectional synthesis

• Result: 2 identical DNA molecules, each composed of a new strand and an old strand

• Chromosome recoils and assumes original shape

In bacteria• Remember: bacterial DNA is supercoiled

which makes this tricky • Replication begins at the origin• 2 replication forks form because bacterial

DNA is circular = bidirectional• Bacterial chromosome is attached to cell

membrane ensuring that each daughter cell receives one copy

• And then…cell wall and membrane form septum and binary fission is complete

DNA replication in bacteria

RNA and protein synthesis

DNAcode

mRNAcodon

transcription translation

Ribosome + tRNA

protein

RNA nucleotides• Same general structure as

DNA nucleotides, except:– Sugar is ribose– RNA does not contain

thymine• RNA nucleotides contain

the following nitrogen bases: – Adenine (A), Guanine (G),

Cytosine (C), and Uracil (U) • mRNA carries the codon =

triplet code• tRNA is the anticodon

Transcription in prokaryotic cells

• Specific gene on chromosome is unzipped• RNA nucleotides from cytoplasm matched to

bases on DNA template using RNA polymerase and beginning at the promoter site.

• This occurs on one side only and follows base pairing rules

• RNA synthesis is in 5’3’ direction• Continues to terminator region• mRNA and RNA polymerase released• mRNA moves to ribosome to begin translation• Chromosome zips back up and returns to

original configuration

Transcription in eukaryotic cells

• Same as prokaryotic, except:• Transcription occurs in the nucleus• Eukaryotic DNA contains introns and

exons• Introns must be removed after

transcription is complete so that the final mRNA contains only exons

• mRNA leaves nucleus through the nuclear pore to the ribosomes in the cytoplasm, where translation occurs

Translation• mRNA associates with

ribosome• Ribosome moves along

mRNA in 5’3’ direction• tRNA brings in amino

acids and matches anticodon to codon using base pairing rules

• Empty tRNA’s return to the cytoplasm to pick up more amino acids

• Growing protein folds into its 3D structure and detaches from ribosome

Regulating gene expression

• Goals– to save energy by only synthesizing

proteins when needed– Try to control rate at which proteins

are synthesized by targeting mRNA synthesis

– Accomplish these goals by repression and induction at operons = special sites which regulate gene expression

Repression and induction

• Repression– Repressors block the ability of RNA

polymerase to initiate transcription of a particular gene due to overabundance of product

– Repressors are regulatory proteins• Induction

– Transcription of gene is triggered by presence of an inducer

– Production of inducer may be triggered by presence of a certain product

• Ex) presence of lactose in E. coli triggers lactose metabolism

Lac O = operator = binding site for repressorLac P = promoter = binding site for RNA polymerase, gene encoding repressor proteinLac I = inhibitor = binds to DNA at operator & blocks binding of RNA polymerase to promoterPi = promoter for Lac ILac Z = codes for beta-galactosidase which hydrolyzes bond between glucose & galactoseLac Y = codes for lactose permease, integral protein carrier for lactoseLac A = thiogalactoside transacetylase = unknown function

Lac Operon

No lactose present Lactose present

= inducer = lactose

**In absence of lactose, repressor binds to DNA and blocks RNA polymerase so that it can’t get beyond promoter

Genetic diversity

• Mutation– Change in the DNA base sequence– Three possible results

• No change (neutral)• Harmful change• Beneficial change

• Genetic transfer and recombination– Genetic recombination– Bacterial gene transfer

Mutations• Two major mechanisms

– Spontaneous mutations = error during replication

– Mutagen = environmental agent induces change directly or indirectly (radiation, chemicals)

• Two major types– Point mutation

• One base is substituted for another– Frameshift mutation

• Deletion or insertion of one or more nucleotide pairs in DNA

Genetic recombination• Exchange of genes between 2 DNA

molecules to form new combinations in chromosome

• Eukaryotes = sexual reproduction• Prokaryotes =

transformation/conjugation– These are very infrequent

Bacterial transformation• DNA is free in the environment• Transferred from one bacterium to

another – Cell that receives new DNA =

recombinant– Cell that gives up the DNA = donor– Cells that can take up DNA = competent

cells• Griffith’s experiment with

Streptococcus pneumonia

Griffith’s experiment: bacterial transformation

Encapsulated S. pneumoniae

+=

+

+

+

Unencapsulated S. pneumoniae=

=

=

Heat killed encapsulated

Heat killed encapsulated + live nonencapsulated

What was responsible?• Avery, Macleod, & McCarty

demonstrated DNA was responsible 20 years later

• Changes in cell wall make it possible for cells to uptake DNA– Not all cells do this naturally– Can be induced

• high concentrations of Ca++

• Electroporation = exposure of cell to pulsed electrical fields induces pores through which cells can accept plasmids or through which plasmids can be removed for donation to other cells

Bacterial conjugation• Involves cell to cell contact & conjugative

plasmid in donor cell• Conjugating cells must be of opposite

mating type• Recipient cell usually does not carry

plasmid• Single stranded DNA transferred to

recipient in which complementary strand is synthesized

• May remain as plasmid in recipient or be incorporated into chromosome– Gram negative = produce sex pili– Gram positive = connect due to sticky surface

molecules

Bacterial conjugation

Transduction

• Donor DNA transferred by bacteriophage• Not all can transduce or be transduced• Generalized transduction

– Host DNA from any part of genome becomes part of DNA of mature virus particle replacing viral genome

• Specialized transduction– Integration of DNA from host chromosome into

viral genome replacing only portion of viral genes

Plasmids• Found in both eukaryotic and prokaryotic

cells• In prokaryotes

– Circular, self-replicating pieces of DNA, usually not essential

– Dissimilation plasmids• Contain genes coding for enzymes that catalyze

unusual substances • Enhanced pathogenicity

– Resistance factors (R factors)• Confer antibiotic resistance, resistance to heavy

metals & cellular toxins

Transposons

• Eukaryotic and prokaryotic• Jumping genes• Very rare, evolutionary significance

– One part of chromosome to another– To another chromosome or plasmid– Can be carried between cells on plasmids

or viruses

Recombinant DNA• Any manipulation of genes• Within a particular species or between

species• Create gene factories

– Insert DNA from another organism into bacterium which subsequently expresses gene

– Can also be used for amplification– Easy to work with because bacterial DNA is

haploid!• Genetic engineering = Biotechnology

Why does this work?• Bacterial defense mechanism against

viral DNA = restriction endonucleases– Enzymes produced by bacteria which cut

unmethylated DNA at specific sites• Digest = piece of viral DNA cut by

restriction enzyme• Palindrome = DNA sequence that reads

from right to left or left to right

GAATTCCTTAAG

Recombinant plasmid• Cut can produce “sticky ends” = will

aneal to complementary stretches of DNA

• If cut DNA sequence to be inserted with same restriction enzyme, H-bonds will form and DNA ligase can insert them and covalently link them so that you have a recombinant product

Recombinant Plasmid

E. coli transformed with GFP