Microbiology 2421 Lecture Notes Microbial Genetics and BiotechnologyDr

Microbiology 2421 Lecture Notes
Microbial Genetics and Biotechnology
Dr. Weis

Definitions:

Genetics: the study of the science of heredity

Genome: all the genetic information in a cell (or virus)

Genomics: sequencing and characteristics of genome

Chromosomes: DNA structures that carry hereditary information

Genes: sections or segments of DNA nucleotide sequences that code for functional products such as RNA (which in turn are used to make a polypeptide that could be an enzyme).

Strain: cloned genetic material used by a geneticist

Genotype: genetic makeup of an organism that codes for its characteristics

Phenotype: expressed manifestation of the genotype, primarily the sum of its proteins.

Genetic Code: set of rules that determines how a nucleotide sequence is converted to a functional product, such as a polypeptide.

Mutation: permanent alteration in chromosome by changing the DNA

Recombination: alteration in chromosome by acquisition and incorporation of new DNA from another organism.

Locus: site of genetic activity

DNA Structure:

Nucleotide macromolecule

Nucleotide = N-base + 5C sugar + Phosphate

Sugar + Phosphate form the backbone, N-Base Hydrogen bond

2 stranded helix

Base pairing rules: purine to pyrimadine. A<-> T and G<->C

Bacteria have a single circular chromosome and associated proteins, looped to create domaina and supercoiled and attached at several points to theplasma membrane and is located in the nucleoid region. Haploid

Replicates just prior to binary fission

RNA Structure:

Nucleotide macromolecule

Nucleotide = N-base + 5 C sugar + Phosphate

Sugar + Phosphate form the backbone, N-base form Hydrogen bonds

Single strand, folds to form the three types: mRNA, tRNA, rRNA

Base pairing rules

DNA to RNA A <=> U, C<=>G, T <=>A

RNA to RNA A<=>U, C<=>G

Enzymes:

Substances, usually proteins, that speed up the rate of chemical reaction in the cell

Since they lower the activation energy, the energy that must be supplied in order for molecules to react with one another.

Apoenzyme (protein portion) + cofactor (nonprotein portion) = holoenzyme

Cofactors = non organic (ions), coenzymes = organic (coA, NAD)

Enzymes are not used up in the reaction and are highly specific.

Examples:

DNA gyrase: helps to supercoil the DNA after folding

DNA polymerase: helps to add nucleotides during DNA replication in 5’ to 3’

RNA polymerase: helps to base pair RNA N-bases to DNA N-bases

DNA ligase: joins Okazaki fragments together to form a complimentary DNA in the 3’ to 5’ direction

Optimum environment: temperature, pH, salt conditions.

Changes cause denature.

DNA Replication

A> Semiconservative (new strand with old/parent strand)

ATP required

Unwinds/uncoils via DNA helicases and stabilized via Helix destabilizing proteins so that the two strands cannot rejoin while the copies are forming.

Hydrogen bonds break

Replication fork: point at which replication occurs is origin of replication

Replication can be bi-directional, creates a “Y” shaped replication fork

Leading strand synthesized continuously by DNA polymerase (I) in 5’->3’

Lagging strand is discontinous and uses RNA primer first to add complementary RNA nucleotides to create the RNA primer.

Then DNA polymerases replace (III) and digest (II) the RNA primer.

After the fragments of DNA are synthesized they are then joined together by DNA ligase.

Hydrogen bonds form between the "old" and "new" strands

After replication, each copy binds to plasma membrane at opposite poles

B> Rolling Mechanism

Seen during conjugation – mating process

One strand breaks away from the other and rolls off the loop.

The other strand remains closed and in a loop.

Both serve as templates for the new daughter strand that forms with each.

Plasmids

2% of genetic information, ~ 5-100 genes

double stranded, circular, extra chromosomal DNA

not essential for normal bacterial growth

independently multiply

circular units contain information about selective advantages since it codes for proteins not coded for by the nucleoid

R-factors => resistance to AB

Bacteriocins => toxins for other bacteria

RNA:

Transcription

RNA polymerase binds to site on the promoter region on DNA template strand

Copies in 5’ -> 3’ direction to create mRNA

Assembles free nucleotides matching N-Bases

Stops when reaches a terminator sequence on the DNA and releases mRNA

Translation

mRNA codons are read by rRNA in a 5’->3’ direction

begins at Start codon and ends at Stop (non-sense) codon

Start codon AUG for bacteria is formylmethione (modified methionine)

tRNA anticodon is matched to the mRNA codon at the P site.

The next tRNA moves into the A site of the ribosome

AA acids brought by the tRNA are joined by peptide bonds via dehydration synthesis from ribozyme in the 50S subunit of the ribosome.

Because mRNA is produced in the cytoplasm in bacteria, transcription and transclation can occur simultaneously. No introns are contained in the mRNA, only exons, the copy of the DNA that codes for expressible genes.

Regulation of Bacterial Gene Expression

* Constitutive: genes always on, not regulated, @ fixed rate

seen for major life processes such as glycolysis

60-80% of genes are constitutive

* Regulated: genes on for transcription/translation, only when needed

Regulation of transcription and translation = Regulation of enzymes for these processes

These enzymes can be controlled or regulated two ways:

1) Controlling synthesis/formation (genetic control)

2) Controlling the activity of the enzyme (feedback inhibition)

Genetic Control of enzyme synthesis/formation

* control transcription of mRNA

* involves induction or repression by regulatory proteins

* regulatory proteins can bind to DNA to block or enhance RNA polymerase function

* regulatory proteins are part of the operon or regulon system

operons : gene sets that are transcribed as a polycistronic unit

regulon: gene sets that are transcribed as a monocystronic unit

* Regulatory proteins may function as repressors or activators

A) Repression

Mediated Regulatory proteins called repressors that block transcription of mRNA

* from binding to promoter region

* from progression down DNA

Inhibits gene expression by usually binding to the operator

Decreases enzyme synthesis, also called negative control

Signal for repression is usually too much metabolic end product

Two types of repressors

a) those that need a co-repressor to be activated

these repressors can not by themselves bind to the operator

the co-repressor binds and alters the repressor shape to a form that can bind to the operator and block transcription.

b) those that are already able to bind to the operator, but can be turned off.

If the active repressor is blocked by another molecule to alter its shape and make it unable to bind, this molecule is called an Inducer since it can turn on transcription of a gene. Resulting products are called inducible enzymes

B) Activators

Regulatory proteins called activators that promote the transcription of mRNA

Normally, activators control genes that have a promoter to which RNA polymerase cannot bind. In addition, the activator protein can not bind to the activator site in its native form.

In an inducer binds to the activator to change its shape and allow it to bind to the activator site. Once this happens, RNA polymerase can bind to the promoter site and begins transcription.

This is called positive control

C) Translational Control

Antisense mRNA is produced (the complementary strand of mRNA)

This complementary strand binds to the mRNA to prevent it from being translated into a protein/enzyme.

Feedback Inhibition (controlling the enzymes activity)

A) Noncompetitive Ihibition

Inhibitor is an end product that binds to the allosteric site on the enzyme. This alters the shape of the active site, thus preventing the binding of the substrates and the pathway is turned off.

B) Competitive Inhibition

Inhibitor is the end product that can bind to the active site to prevent the enzyme from binding to its normal substrate.

Operon Model

Operon = structural genes and their control regions

Parts:

Control Region

a) Promotor = site for RNA polymerase

consists of a recognition site and a binding site for RNA polymerase to unwind DNA begin transcription

b) Operator = go / no go signal for transcription of DNA

Structural Region

Structural genes that code for an end product

(i.e. mRNA for protein synthesis)

Regulatory genes for the operon that occur in front of the operon

Repressor proteins can then signal "stop" by blocking RNA polymerase

Inducers can bind to repressors to allow RNA polymerase to "go"

Thus, based on the previous information on enzyme contro, two types of operon systems are seen:

A) Inducible operons

Inducer can block active repressor so it can’t bind to operator site

Can be a by-product of metabolism or coded from the regulatory genes

e.g. Catabolic pathway for Lactose (in absence of Glucose)

B) Repressible Operons

Inactive Repressible proteins

A Co-repressor binds to an inactive repressor to activate it and allow it to block transcription and translation.

Excess end or by product usually acts as the co-repressor

Therefore, structural genes are "on" until they are repressed (turned "off")

e.g. Anabolic pathway for Tryptophan

Mutations: An error during DNA replication that results in the changed sequencing of DNA bases. Can occur in regulator genes, structural genes, RNA genes, noncoding genes

Types:

* Silent (neutral) : the change in DNA sequence cause no change in product activity. One nucleotide substituted for another

Degeneracy of the code: AA signal has several codons

Wobble effect of binding @ # 3 Nitrogen-base of codon-anticodon

AA substituted has similar chemical properties as original

* Point mutation : base substitution in DNA sequence does cause a change in the activity of the end product.

* Transition: allow for substituting a purine for a purine (A for T and vice versa)or a pyrimidine for pyrimidine (C for G and vice versa)

* Transversion mutations: allow for substituting a purine for a pyrimidine (or vice versa)

* Frameshift mutation: deletion or insertion of one or more nucleotides shifts the reading frame off from normal triplet base pairs that creates inactive protein due to change in AA sequence e.g. Huntington’s Disease (Autosomal Dominant) where many bases added into a gene that causes progressive neurological degeneration in ACh producing neurons.

Mutation Causes:

^Spontaneous Mutations

Usually happens during replication

Occur in absence of mutating causing agents

Types: Transition, Transversion, or Frameshift deletions, replication errors, and loss of the Nitrogen in the nitrogen base.

^Induced Mutations

DNA exposed to mutagens: (chemical or physical)

Chemical Mutagens:

Nitrous acid: changes A to a molecule that base pairs with G, not T

Nucleoside (sugar + N-base) substitutes/analogs: takes place of normal nucleosides unable to base pair (Hydrogen bond) properly

Toxins (smoke, soot, mold) create frameshift mutations

Intercalating agents: push nucleotides apart so extra is added

Physical Mutagens:

X-rays, Gamma Rays: free radicals damage DNA base pairs and prevent repair or break Sugar –Phosphate backbone

UV light: links T to C, does not allow T-A or C-G, Links adjacent T to T to form Thymine dimmers. Length of exposure determines how severe the damage.

^Conditional Mutations

Those that are expressed under certain environmental conditions

* auxotrophs: mutants that cannon grow with minimal requirements

* prototrophs: mutants that can grow with minimal requirements

Directive or adaptive: can choose which mutations occur given the requirements.

^Other "Mutations": Transposable Genetic Elements or "Jumping Genes"

Cut themselves out of DNA or plasmid and can instert into another piece of DNA or plasmid. Use enzymes such as transpoase and integrase.

Types of Transposable Genetic Elements:

Insertion Sequences: small fragments of DNA copies (1-12 genese) that do not contain genetic information, only genes coding for enzymes required for transposition . Have ability to insert segment into chromosome.

Also known as conjugative transposons if part of DNA.

Transposons: larger DNA fragments do carry genetic information.

Also called integrons that can be inserted and accumulate in the plasmid or chromosome.

Contain one or more active genes boarded by repeated inverted base sequences.

Clusters of genes involved are called gene cassettes.

Causes overlapping to create new protein sequences

Mutation Rate

Probability that a gene will mutate when it divides

Spontaneous very low rates 10 to the -9 power, allow for adaptation to environment

Harmful mutation – dies

Beneficial mutation- survives and passes along trait

Mutagen increases rate of mutation by possibly doubling rate.

(If normal mutations rate was 10 to -6, mutagens could cause 10 to -3 power increase in rate)

Expression of Mutations:

Normal genetic make up = “wild type”

Mutation from prevalent gene =”forward mutation”

Mutation causing it to appear normal ="reversion mutation" or "suppressor mutation" if it is a second mutation that masks the first mutation

Mutation that causes return of original genetic makeup ="back mutation"

Mutagen Detection

Altered phenotype selection or testing

* Positive or Direct Selection: reject normal cells, accept abnormal

* Negative or Indirect Selection: selects cells that cannot due function

Using Mutant Bacteria to test to see if a substance is carcinogenic: Ames Test: Reverses bacteria to "normal", therefore number of new normal mutants which equates to the degree of carciinogenicity.

Repair Mechanisms for Mutations

DNA repair

DNA polymerase : proof reads the complimentary strands

Nucleases: enzymes that excise damaged DNA, allow for new DNA to form replacement as complementary strand

DNA ligase: joins DNA fragments together

DNA glycolases: removes damaged or unnatural DNA bases

Rec A protein: use a replicated copy as template to repair "original"

SOS repair: inducible repair, uses recA to help when there is extensive damage to DNA.

Genetic Transfer and Recombination

Genetic Recombination: exchange of genes between two DNA molecules to form a new combination of genes on a chromosome, normally accompanied by a phenotypical change.

e.g. Crossing over: between two related chromosomes (Eukaryotes)

Vertical Gene Transfer

Genes passed from an organism to its offspring

Exogenote from the donor to be incorporated into the endogenote of the recipient.

Horizontal Gene Transfer

Lateral transfer from one microbe to another in the same generation

Transfer involves donor and recipient cell

Can be chromosomal DNA or plasmid

Chromosomal DNA is incorporated and takes the place of the old DNA

Recipient now called a recombinant

If transfer of plasmids, the plasmids function independently

Genetic Recombination Types:

* General Recombination

reciprocal change between homologous DNA sequences

any place on the chromosome

results from DNA strand breakage and reunion

carried out by products of the rec genes (recA)

* Site Specific Recombination

genetic material is not homologous

important in integration of viral genomes into bacterial chromosomes

* Replicative Recombination

replication of genetic material

does not depend on sequence homology

Movement of DNA from a donor bacterium to a recipient takes place three ways:

Transformation, Conjugation, Transduction

Exogenote -> Endogenote via one of three ways (above) => Merozygote (bacteria with temporary diploid genome.

DNA of Exogenote’s fate in the endogenote (recipient)

~ integrated

~ partial diploid clone, exogenote DNA in recipient can replicate

~ partial diploid cell, exogenote DNA in recipient cannot replicate

~ host restriction, exogenote DNA is degraded by cell nucleases

Transformation:

Takes place in less than 1% of bacterial population, 1 cell in 1000

Genes transferred to another in solution or environment

Random process, any portion of the genome may be transferred

Usually seen after lysis of bacteria, releasing DNA fragments (~ 20 genes) into the environment

Dependant on certain conditions:

Certain stage of growth (exponential phase)

Ability to secrete a competence protein

Discovered by Griffith using R (unencapsulated) and S (capsule) strains of Streptococcus pneumoniae

Can occur with plasmids (laboratory setting) or DNA fragments (naturally)

Double stranded DNA fragments or plasmids from donor cells are taken up by recipients-> now called recombinants

Occurs naturally in a few genera

Bacillus, Haemophilus, Neisseria, Acinetobacter, some Streptococcus and Staphylococcus

Pseudomonas, Azobacter, Moraxella

Works best when donor and recipient are closely related, ie. similar DNA and in

Close contact/ crowded conditions.

Alterations in cell wall and plasma membrane make cell "competent" so that one strand of DNA can enter to displace a segment of the recipient’s DNA by means of recA proteins.

The displaced "original" DNA segment is degraded by cellular enzymes.

Competence factors are reflected in an organisms surface receptors for binding DNA, changes in membrane permeability, or sometimes induced by environmental changes.

Purpose: increase organism’s pathogenicity

Conjugation:

Transfer of DNA from a living donor bacterium to recipient @ close contact

G(-) use sex pilus, G(+) use sticky surface

Types of Conjugation: F+, Hfr, Resistance Plasmid.

Mediated by a F (fertility) factor either as a plasmid [F+] or incorporated into the donor chromosomes [Hfr] cell

Direct cell to cell contact necessary for conjugation to occur

Opposite mating types, that is the donor carries the F factor the recipient does not.

Sex pili and sticky surface molecules are normally used, coded for by the plasmid, which consists of 20-30 genes that code for enzymes that replicate DNA and make sex pili. The sex pili form a channel or conjugation bridge between the adjacent cells.

Once contact has been made, the plasmid is replicated via the rolling circle method. During transfer, the single strand copy moves to the recipient and then the complementary strand is replicated in recipient.

For plasmid conjugation, a F+ transfers its plasmid to a F- to make it F+

The single strand plasmid in the donor cell is copied and the helix reforms.

This plasmid can stay separate in the new F+ recombinant recipient or now be integrated into recipient’s DNA at certain sites to create a new Hfr cell

For DNA conjugation, a Hfr transfers part of its chromosome to a F- cell.

Usually the chromosome breaks before it is completely transferred, a time dependent process. The recipient cell is now a recombinant F-.

Unused plasmid segments are then degraded in the recipient cell.

If plasmid exchanged has genes that code for antibiotic resistance, called a resistance plasmid conjugation. One strand is left in the donor, the other sent to the recipient. After conjugation, both donor and recipient make the complementary copy of the R-plasmid.

Conjugation occurs between various genera

E.coli – Shigella

E.coli – Salmonella

Serratia – Salmonella

Purpose: increase organism’s pathogenicity or virulence

metabolic changes

fertility factor

(sex pili for future conjugation)

Other bacterial genera do not make sex pili, but clumping factors that allow close proximity of cells so that pores can form between the species.

e.g. Bacteroides, Clostridium, certain Strep spp.

Transduction

Transfer of bacterial genes by viruses

Bacterial DNA is transferred from a donor to recipient via a virus that infects the bacteria, called a bacteriophage or phage. For this process, they are called transduction phages.

Two types of transduction: Generalized and Specialized

Generalized Transduction

Phage attaches to bacterial cell

Injects DNA into bacteria which acts as template for new phage and protein capsule coats (capsid)

Phage enzymes break @ bacterial DNA and some of it is incorporated into phage protien capsids, so that phage DNA carries bacterial DNA instead of phage DNA. Quantitiy of bacterial DNA depends on size of capsid

Released phage can infect another bacteria and transfer bacterial genes

If bacterial phage DNA is inserted it is unable to initiate lytic phase

If phage DNA is not incorporated into bacteria, called abortive transduction and the bacteria are considered partial diploids.

Specialized (Restricted) Transduction

Only certain bacterial genes are transferred along with phage DNA.

Error in lysogenic life cycle due to abnormal excision of phage DNA

Phage can code for certain toxins produced by their bacterial hosts

Defective phage cannot reproduce, but can inject bacterial genes into another bacterium, so that it contains new bacterial DNA and viral DNA.

Replication cycle of the bacteriophage occurs one of two ways

Lytic phase: virulent phages infect bacteria, replicate and lyse bacteria. Usually associated with generalized transduction

Lysogenic: temperate phages infect bacteria, replicates at later time. Cells appear normal even with viral replication. Incorporated viral genome is called a prophage. Usually seen in specialized transduction

BIOTECHNOLOGY and Recombinant DNA

Genetic Engineering:

Inserting genes of interest into bacterial DNA

Allow recombinant vector to grown to form clones, identical copies

Enzymes used:

Endonucleases: enzymes that cleave sugar-phosphate bonds

Restriction Enzymes: act at particular sequences of nucleotide bases

Usually 4, 6, or 8 base pairs long with staggered "sticky" ends

DNA ligase: used to rejoin DNA pieces

Reverse transcriptase: produce DNA copies from RNA genome

Selection of bacteria to use:

Natural selection

Artificial selection

Self directed mutagenesis

DNA sources: Lyse cells and precipitate DNA; Phage Gene library; Reverse transciptase of mRNA to make cDNA [c = copy]; synthetic DNA

Vectors:

Characteristics

1) capable of replicating

2) size for manipulation (usually small)

3) Preservation: circular form, prevent destruction

4) Code for a particular phenotypic trait

Examples of vectors used

1) Plasmids

2) Shuttle vetors: plasmids inserted into various organisms

3) Viral DNA / bacteriophages

4) Artificial chromosomes

Vector + genes of interest > Recombinant bacteria that is cloned to make:

a) gene copies which can be inserted (into plants or bacteria)

b) protein product (vaccines, hormones, immune chemicals)

c) Amplify DNA to be used for analysis and experiments