Chapter 8

Bacterial Genetics

Bacteria are a good model system to use to study genetics

A Genetic change is something that changes the organism’s genotype

  • genotype-sequence of nucleotides in an organism’s DNA

some genetic changes are caused in observable characteristics or phenotype

Since bacteria are haploid, genotypic change normally results in phenotype change and can be drastic

Haploid - one copy

Genetic Change caused by mutations and horizontal gene transfer

  • mutations-vertical transfer, single cell passed onto progeny

  • Gene transfer-Horizontal /lateral. gene acquired from another cell and then passed on

mutants can have phenotype changes

  • Autotrophs - require a growth factor

  • prototrophs - can grow without growth factor

  • cell resistant to microbial agent

  • cell senistive to microbial agend

Indirect Selection

spontaneous mutations

occur naturally and randomly

Genes have different rates of spontaneous mutation.

  • the probability that a mutation will occur in a given gene each time a cell divides

    • mutation rate

      • low rates: 104 510 12

occur independently of one another

  • Treated with 2 drugs

mutations are usually stable but can sometimes change back to the original form

  • reversion

passed on to progeny

The environment does not cause mutations

  • Affect whether mutation remains in a given population

Base Substitution

most common.

Incorrect base incorporated during DNA synthesis

Point mutation - single point change

If a mutation results in a new codon encoding for a different and it is a missense mutation

  • leaky - protein is partially functional

nonsense mutation - stop codon mutation

  • the new protein is truncated

silent mutation - If the mutation does not result in an amino acid change (new codon same as original)

  • does not alter the protein product

Any mutation that inactivates the gene resulting proton completely is null or knockout

The rate of base substitutions can be increased by oxygen in the environments

  • reactive forms of oxygen can oxidize guanine which is usually then mispaired with adenine

Add/Remove Nucleotides

This happens in the normal course of DNA replication

Add/remove in sets of 3 adds/removes an

Add I remove one or two results in Shift mutation

Induced Mutations

study of mutations is essential for research

mutagens help us by inducing mutations

  • Increase the 1D00X

Mutagens

  • Chemical

  • Reduction

Transposition

Repair DNA Damage

DNA is continuously damaged under natural environmental conditions

mutation rates are so low because most of that DNA damage is repaired

repair errors in Base incorporation

repair modified Bases

50s Repair

Mismatched Repair

Fixes what polymerase doesn't catch

mismatch results in incorrect DMA Structure

  • bulge

Involves Cleavage, excision of the mispaired region

  • filled correctly

new strand lacks methylation

Direct Selection

mutant cells grow, parental cells do not

Antibiotic resistance mutations

Indirect Selection

more complicated, often have to look for or select for a lack of growth

often used to isolate the auxotrophic parent populations

methods

  • Replica Plating

  • penicillin enrichment

Replica Plating

master plate with mutants and wild-type combines

Transfer onto Steril velvet

Allows transfer of colonies to different plates

  • exact copies

one plate with enriched media and one with minimal media

orientation is important

Penicillin Enrichment

Increases the ratio of mutants to nonmutants in a population

  • not by creating more mutants but not getting rid of some nonmutants

Penicillin targets actively growing cells

Auxotrophs growing in minimal media are not actively growing

Penicillinase enzyme destroys penicillin

plating onto enriched medium albus growth of mutants and any non-mutants still alive

Replica plating can distinguish between the rest

Horizontal Gene Transfer

When microorganisms acquire genes from other cells

Transfer methods

  • Transformation

  • Transduction

  • conjugation

DNA-Mediated Transformation

uptake of DNA into a cell.

  • DNA contained in a cent or virus (Naked DNA)

In nature, naked DNA can come from lysed cans and some bacteria secrete small prices of it

The recipient cell must be competent to take up the DNA

  • Artificial competence

  • Natural competence

DNA must be part of a replication to be maintained in a population

Natural competence

a physiological condition that varies between species

  • some bacteria is always competent some only at the critical population

Sometimes only about 10% of the population become competent

some competent bacteria accept all DNA and some only from related species

Transformation

ds DNA binds to receptors on competent cell

only one strand emerges, nucleases degrade the rest

Donor DNA integrates into a homologous region the recipient genome by homologous recombination

In the lab, DNA confers a selectable marker like antibiotic resistance

Artificial competence

Allows introduction of ds DNA into most cells even if they are not naturally competent

Electroporation-electric current makes holes in a cell wall/ membrane allowing the take-up of DNA

Transduction

Bacteriophages (Phages) - bacteria viruses

can transfer genes between bacterin cells - Transduction

Phages - nucleic acid surrounded by a proton coat

Attach to bacterial cells, and inject their nucleic acids which encode for enzymes that degrade host DNA

Host cell then a phage factory - then lysis

sometimes, bacteria are included in the new phages produced and released by lysis (Transducting particle)

Generalized Transduction - any genes from a donor can be transferred

Specialized Transduction - only a few specific genes transferred

Conjugation

Transfer of genetic information between bacteria cells by direct contact using Sex pilus (F-Pilus)

occurs in Gram pos. and Gram neg

Plasmids and portions of the chromosomes transfer

Plasmid Transfer

most frequently transferred by conjugation

Conjugative Plasmids

plasmids with specific genes that facilitate transfer by conjugation

direct own transfer from donor to recipient cells

The most studied example is the F plasmid of E. Coli

Plasmids contain genes for antibiotic resistance or other things that may contribute to an organism’s fitness

Conjugation transfer of F Plasmid

Donor cell F plasmid contains genes to synthesize f pilus and endonuclease enzyme

Step 1

  • F+ cell binds a receptor on the acceptor cell and pulls it in

Step 2

  • f+ cell endonuclease cleaves one strand of f plasmid at the origin of transfer.

  • Plasmid now ssDNA with endonuclease at one end

Step 3

  • Ss F plasmid

    • Apr. 2 Min.

Step 4

  • Complementary strands synthesized for 55 plasmids in both cells

  • both cells now F+

Mobil Genetic elements

DNA that can move around the genome

  • plasmids

  • Transposons

  • Genetic islands

  • other things like bacteriophage elements and insertional sequences

Plasmids

In Most Bacteria and Archaea organisms as well as many Eukarya

most double-stranded, circular DNA, can be replicated

Does not encode any of the core genome or any normally essential genes

few genes to thousands

Low copy number plasmids - few copies per cell

High copy number plasmids - marry espies per cell

most plasmids have a narrow host range and can only replicate in one species

  • few have broad host range

many transferred by conjugation

Resistance Plasmids

Provide the organism with resistance to antimicrobial medications and heavy metals

Has resistance conferring genes

  • needed for conjugation

The broad host range for gram-negative bacteria

spontaneous mutations

Transposable elements

  • classic studies carried out by Barbara McClintock

  • observed color variation in corn resulting from transposons moving in and out of genes strolling pigment synthesis

Transposons

Simplest - insertion sequence encodes only transposase

composite transposon encode for at least one additional

gene

  • easily recognized

    • non-homologes replication

Transposable Elements

Transposons - jumping genes

set of genes that can insert into or remove itself from PNA-Chromosome or plasmid

If inserts into and disrupts a gene-Insertional inactivation

have transcriptional terminators so they can disrupt downstream genes if in an polycistronic message

Induced Mutations

Transposon

introduce transposon into cells to induce mutations

inserts into the genome in order to be replicated

gene inserts into is activated usuary rendering it a knockout mutation

Chemical modification of bases

Chemicals modify purines and Pyrimidines

Alkylating agents - reactive chemicals add an alkyl group to purines and pyramides disrupting hydrogen bonds

Nitrous Acid converts the amine Group to the keto group Which Changes nucleotide identity and subsequent pairing

Chemical mutagens

Base Analogs

  • resemble purines and pyridines are mistakenly incorporated

Intercalating Agents

  • flat molecules of similar size to the nucleotide and can fit in between the bases

  • causes errors during replication

  • often resulting in frameshift mutations and/ or premature stop codons

  • Ethidium Bromide

Radiation

UV

  • two thymines next to each other form a covalent bond

  • dimers don't fit into the helix properly

    • cannot be separated or used as a template for replication

      • cause cell death

  • mutations arise in the methods used to repair damage

X-rays

  • Sin