Microbial Genetics.

Microbial Genetics

Definitions Genetics Genome
the study of heredity, genes and the mechanisms that they carry this information Replication Expression Genome Complete genetic information of the cell

Definitions Chromosome Gene Genomics
The structures that are composed of DNA that carry the hereditary information Gene Segments of the chromosome that code for a specific product (usually a protein) Genomics Sequencing and molecular characterization of genomes

Definitions DNA (deoxyribose nucleic acid) Nucleotides 3 components
Phosphate Deoxyribose sugar Nitrogenous base Adenine, thiamine, cytosine or guanine Double helix (complementary strands) Base pairs A-T C-G A-U (RNA) Hydrogen bonds

DNA Base sequence codes for protein 4 letter alphabet (A, T, G and C)
Genetic code Determines how nucleotide sequence is converted into amino acid sequences Complementary strand allow precise duplication

DNA to proteins Gene on DNA Converted to mRNA mRNA on ribosome
tRNA brings amino acids to ribosome for protein synthesis

Definitions Genotype Phenotype Genetic information of the organism
Information that codes for characteristics of the organism Phenotype The expressed or physical characteristics of the organism The expression of the genotype

Bacterial Chromosome (DNA)
Single Circular Attached one or many sites to plasma membrane

Bacteria chromosome Escherichia coli 4.6 million base pairs 4300 genes
1mm long 1,000 X length of cell Supercoiled Topoisomerase II DNA gyrase

Bacterial chromosome Genetic map Mapped in minutes
Based on time for chromosome exchanged between two cells

DNA replication Parental strand Two new “daughter strands”
Each strand acts as template for new strands Semiconservative replication

DNA Replication Carbons in nucleotide numbered 1`-5`
Complementary sugars are upside down to one another Strands run 5`3` on each side

DNA Replication Steps in replication DNA unwinds DNA polymerase
Adds nucleotides to 3` end Replication fork forms Leading strand forms towards the fork 5`3`

DNA Replication DNA replication Lagging strand Needs RNA primer
Removed by DNA polymerase Synthesized discontinuously Moves away from fork Okazaki fragments 1000 nucleotides DNA ligase fuses segments

Bacterial DNA Replication
E. coli Occurs bidirectionally Two replication forks Continues until forks meet

RNA Synthesis Transcription Translation
Process of taking DNA code and converting to RNA code Translation Converting RNA (mRNA) with tRNA to form amino acid sequences and proteins Occurs at ribosome

Protein Synthesis Three types of RNA DNA unzips at gene
mRNA - messenger tRNA - transfer rRNA – ribosomal DNA unzips at gene

Transcription RNA polymerase binds to DNA at promoter
Only coding strand of DNA is template 5`3` direction RNA polymerase assembles RNA nucleotides

Transcription RNA chain grows RNA stops growing at terminator site
mRNA strand released from DNA DNA zips up mRNA intermediate between DNA and translation

Translation Bacterial translation Protein synthesis
Decoding mRNA to amino acids and proteins Codons Groups of 3 nucleotides Sequence of codons determines amino acid sequence Several codons for a single amino acid Degeneracy Allows for mutations

Translation 64 codons (43) Sense codons Nonsense codons AUG
Code for amino acids 61 codons Nonsense codons Stop codons UAG, UAA, UGA Signal end of protein synthesis AUG Start codon Formylmethionine Usually removed from protein

Translation tRNA Transfer RNA Anticodon Amino acid attached
Complementary to codon Amino acid attached Brings amino acid to ribosome

Translation 1 – components needed come together
Ribosome tRNA mRNA 2 – tRNA carries first amino acid ( ?) to ribosome and mRNA

Translation 3 – second amino acid brought to ribosome P – site
Site of first amino acid A – site Site of second amino acid Peptide bond forms

Translation 4 – after peptide bond first tRNA is released to find amino acid

Translation 5 – ribosome moves along mRNA until tRNA is in P site
Process continues down mRNA

Translation 6 – ribosome continues down mRNA Peptide chain elongates

Translation 7- polypeptide (protein) released
Ribosome moves down mRNA until stop codon UAG, UAA, UGA Polypeptide released

Translation 8 – tRNA is released and ribosome disassembles
tRNA, mRNA, and ribosome can be used again

Review

Other points Ribosome moves 5`3` direction
Additional ribosome may attach and begin synthesizing protein Prokaryotes can start translation before transcription is complete

Eukaryotic differences
Transcription takes place in nucleus mRNA completed prior to entry in cytoplasm Exons – Expressed DNA, code for protein Introns – intervening DNA, do not code for protein Removed by ribozymes

Regulation of Bacterial Gene Expression
All metabolic reactions are catalyzed by enzymes (proteins) Feedback inhibition stops a cell from performing unneeded chemical reactions Stops enzymes that are already synthesized What prevents synthesis of enzymes that are not needed?

Protein synthesis requires tremendous energy Cell does not waste energy Regulating protein synthesis economizes cells energy

Genes 60-80% are constitutive Not regulated Products produced at fixed rate Genes turned on all the time Code for enzymes essential to major life processes Enzymes needed for glycolysis

Genes Inducible genes Production of enzymes is regulated Inducible enzymes Present only when needed Trypanosoma Surface glycoproteins Produces one glycoprotein at a time Eludes immune system

Regulation of transcription Repression Decreases gene expression Decrease enzyme synthesis Response to overabundance of an end product Regulatory proteins called repressors Block RNA polymerase

Regulation of transcription Induction Turns on genes Substance that turns on gene Inducer Inducible enzymes

Induction enzymes β-galactosidase (E. coli) Cleaves lactose Medium without lactose = little to no β-galactosidase Lactose added to medium large amounts of β-galactosidase produced Lactose is converted to allolactose Allolactose is the inducer Enzyme reduction

Operon Model Three genes for lactose utilization
Located next to each other on bacterial chromosome Regulated together Called structural genes lac structural enzymes are transcribed and translated lac for lactose

Operon Model Operon model lac operon Promoter region Operator region
Region of DNA where RNA polymerase initiates transcription Operator region Go or stop signal for transcription of the structural genes Structural genes Genes for metabolism of lactose

Operon Model Inducible operon Near lac operon is regulatory gene
I gene Codes for repressor protein

Operon Model Lactose is absent Repressor binds to operator site
RNA polymerase is inhibited No transcription of structural genes No mRNA No enzymes are synthesized

Operon Model Lactose is present Converted to allolactose
Inducer Inducer binds to receptor protein Receptor protein altered Does not fit into operator site RNA polymerase is not inhibited Structural genes are transcribed to mRNA then translated into enzymes An inducible operon

Operon Model Repressible operon Tryptophan synthesis
EDCBA structural genes Also has promoter and operator region

Operon Model Repressible operon
Structural genes transcribed and translated Tryptophan is synthesized

Operon Model Repressible operon Excessive tryptophan accumulates
Tryptophan acts as corepressor Corepressor binds to repressor protein Repressor protein binds operator and structural genes no longer transcribed

Lactose regulation Lactose operon
Depends on level of glucose in medium Enzymes for glucose metabolism are constitutive When glucose is absent cAMP (cyclic AMP) accumulates in cell cAMP binds to cAMP receptor protein (CRP) This binds to lac promoter Initiates transcription by allowing mRNA polymerase to bind to the promoter Transcription of lac operon requires Presence of lactose Absence of glucose cAMP is an alarmone Chemical alarm signal the cell uses to respond to environmental or nutritional stress

lac operon

Lac operon Catabolite repression
Inhibition of the metabolism of other carbon sources by glucose Glucose effect

Mutation Mutation Change in the base sequence of DNA
may cause change in the product coded by the gene Beneficial Lethal Neutral Occur commonly Degeneracy

Mutations Types of mutations Base substitution (point mutation)
AT substituted for CG mRNA carries incorrect base Translation Insertion of incorrect amino acid into protein Missense mutation, nonsense mutation, frame shift mutation, and spontaneous mutations

Base substitution

Mutations Normal No mutations DNA strand properly transcribed by mRNA
Correct sequence of amino acids for protein

Mutations Mis sense mutation
Base substitution results in an amino acid substitution in protein Sickle cell anemia A to T Glutamic acid to valine Hb shape changed during low oxygen

Mutations Non sense mutation
Base substitution creates a nonsense or stop codon Protein is not produced Only a fragment of protein is produced

Mutations Frame shift mutation
One or a few nucleotide pairs are deleted or inserted in the DNA Shifts the translation reading frame Almost always result in a long stretch of altered amino acids Inactive protein

Mutations Insertion of extra bases into a gene Spontaneous mutations
Huntington's disease Spontaneous mutations Occur occasionally in DNA replication Mutagens Chemically of physically alters DNA and effects a change is called a mutagen Radiation, ultraviolet light

Mutagens Chemical Mutagens Nitrous acid
Converts adenine (A) to a form that doesn’t bind with thymine (T), but instead binds with cytosine (C) Alters base pair on DNA, works on random locations

Mutagens Chemical mutagens (cont) Nucleoside analogs
Structurally similar to normal nitrogenous bases 2 - aminopurine Adenine 5 – bromouracil Thymine analog Will bind with guanine

Mutagens Chemical mutagens (cont)
During replication analogs cause base pairing mistakes Antiviral and antitumor drugs AZT (azidothymidine)

Mutagens Chemical mutagens (cont)
Other chemicals cause deletions, frameshifts, or insertions Benzyprene – present in smoke and soot Frameshift Aflatoxin – Aspergillus flavus

Mutagens Radiation mutagens X – rays Gamma rays Ultraviolet
Forms covalent bond between certain bases Thymine dimers Death of damage to cell Light repair enzymes Photolyases Use visible light energy to separate dimer

Mutagens Ultraviolet damage Nucleotide excision repair
Enzymes cut out distorted thymines Creates gap Gap is filled with newly synthesized DNA DNA ligase joins strand to surrounding backbone

Mutation frequency Mutation rate
Probability that a gene will mutate when a cell divides Expressed in power of 10 10-4 mutation rate (1 in 10,000 chance of mutation) 10-6 ( 1 in 1,000,000) Mutagens Increase spontaneous mutation by 10 – 10,000 times 10-6 becomes 10-3 to 10-5

Identifying Mutants Positive (direct) selection
Detection of mutant cells by rejection of unmutated parent cells Penicillin in agar Unmutated parental cell will not grow Only mutated cells grow

Identifying Mutants Negative (indirect) selection
Replica plating technique

Replica Plating

Replica plating Auxotroph
A mutant microorganism having a nutritional requirement that is absent in the parent.

Identifying Chemical Carcinogens
A substance found to cause cancer in animals Often mutagens are carcinogens as well Previously used animal testing Time consuming Expensive

Ames test Ames test utilizes bacteria to act as carcinogen indicator
Based on observation that exposure to mutant bacteria to mutagenic substance may reverse effect of the original mutation

Ames test These are called reversions
Back mutations Measures the reversion of Salmonella Auxotrophs Have lost there ability to synthesize histidine (his-) (his+) bacteria have ability to synthesize histidine 90% of substances that cause reversion have been shown to be carcinogens

Ames Test

Genetic Transfer and Recombination
Genetic recombination Exchange of genes between two DNA molecules to form new combinations of genes on a chromosome Crossing over Two chromosomes break and rejoin Adds to genetic diversity

Genetic transfer and recombination
Eukaryotes Meiosis Prophase I Prokaryotes Numerous different ways

Genetic Transfer and Recombination
Vertical gene transfer Genetic information passed from an organism to its offspring Plants and animals Horizontal gene transfer Bacteria transfer genetic information form one organism to another in the same generation Genetic information passed laterally

Horizontal Gene Transfer
Donor cell Organism gives up its entire DNA Part goes to recipient cell Part is degraded by cellular enzymes Recipient cell Receives portion of donor cells DNA Incorporates donor DNA into its own DNA Recombinant DNA Less than 1 % of population

Transformation Genes transferred from one bacterium to another in solution Naked DNA Discovered by Griffith Used Streptococcus pneumoniae Two strains Virulent (pathologic) strain Had a polysaccharide capsule resists phagocytosis Avirulent (non- pathogenic) strain Lacked a capsule

Griffith’s Experiment

Transformation Bacteria after cell death and lysis could release DNA into environment Recipient cell can take up DNA fragments and incorporate into their own DNA Resulting in a hybrid (recombinant cell) Recombinant cell must be competent Able to alter cell wall to allow DNA (large molecule) to enter Bacillus, Haemophilus, Neisseria, Acinetobacter, and some Staph and Strep

Genetic Transformation

Conjugation Conjugation Involves plasmid Requires cell to cell contact
Circular piece of DNA Replicates independent of chromosome Non essential for growth genes Requires cell to cell contact Opposite mating type Donor cell carries plasmid Recipient cell lacks plasmid

Conjugation Gram positive Gram negative
Sticky surfaces cause bacteria to come in contact with one another Gram negative Utilize sex pili

Conjugation E coli model F factor plasmid
Fertility factor Donors (F+) Recipients (F-) Converted to (F+) F+ factor integrated into chromosome Becomes Hfr (high frequency of recombination) cell

Bacterial Conjugation
Hfr conjugates with F- cell Chromosomal strand replicates and transferred to recipient Incomplete transfer of donor DNA Recipient integrates new DNA Acquires new versions of chromosome Remains F- cell

Conjugation in E. coli

Conjugation Minutes and conjugation
Identify locations of various genes Hfr His, pro, thr, leu, and F (+) F(-) His, pro, thr, leu, and F(-)

Transduction in Bacteria
Transfer of bacterial DNA transferred via bacteriophage Bacteriophage Virus that infects bacteria

Transduction Steps of transduction
1- bacteriophage infects donor bacterial cell 2- Phage DNA and proteins, and bacterial chromosome is broken into pieces

Transduction Steps of transduction
3- during phage reassembly, bacterial DNA incorporated in capsid of bacteriophage 4 – donor cell lysis releasing new bacteriophage particles

Transduction Steps in transduction
5- phage carrying donor DNA infects new recipient cell 6- recombination can occur Producing bacteria with genotype different than donor and recipient

Transduction Generalized transduction Specialized transduction
Previously explained Specialized transduction Only certain genes are transferred i.e. phage codes for toxins to be produced Cornybacterium diphtheriae – diphtheria toxin Streptococcus pyogenes – erythrogenic toxin Escherichia coli – Shiga toxin (hemorrhagic diarrhea)

Plasmids Plasmids Self replicating rings of DNA
1-5% size of chromosomal DNA Non – essential genes Conjugative plasmid F factor Dissimilation plasmids Code for enzymes to breakdown unusual sugars and hydrocarbons Help in survival of unusual environments

Plasmids Other plasmids Toxins (Anthrax, tetanus, Staph)
Bacterial attachment Bacteriocins Toxic proteins that kill other bacteria Resistance factors (R factors) Resistance to antibiotics, heavy metals, cellular toxins

Plasmids Resistance factors
Two groups RTF – resistance transfer factor Includes genes for plasmid replication and conjugation r-determinant Resistance genes Codes for production of enzymes that inactivate drugs or toxic substances Bacteria can conjugate and transfer plasmids between species Neisseria Penicillinase resists penicillin

R factor Plasmids

Transpoons Transpoons
Small segments of DNA that move from one region to another 700-40,000 base pairs Occur in all organisms Can insert within genes Disrupt transcription of gene Occurs rarely (similar to spontaneous mutation rate)

Transpoons Transpoons Contain gene for transposition
Insertion sequence (IS) Codes for transposase Cuts and seals DNA for transpoons

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Microbial Genetics.

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