MICROBIO470 Exam 3 Terms

essential genes

• Genes the cell requires to survive and propagate

• Genes for polymerases, replication machinery, etc.

minimal media

• Defined medium containing only a carbon source, phosphate, nitrogen and salts

prototroph

• Strain that can grow on minimal medium

auxotroph

• Strain that requires a specific nutrient to grow

catabolic mutant

• catabolic process is disrupted

vertical inheritance

• Transfer of DNA from an organism to its progeny solely through reproduction

• Vertical gene transfer

horizontal inheritance

• Transfer of DNA between individuals in the population rather than by inheritance from ancestors

• Horizontal gene transfer

• Genes conferring antimicrobial resistance can be spread

• NOT reproduction

transformation

• Uptake of external DNA originating from a donor cell into a recipient cell

• No cell-to-cell contact required, just DNA+ recipient cell

• Cell that is transformed= competent

• Mechanism of HGT

transduction

• Transfer of DNA from one cell to another by a phage

• Mechanims of HGT

conjugation

• Transfer of DNA directly from one cell to another through mating

• Via plasmids

• Mechanism of HGT

antimicrobials

• Medications that are used to prevent and treat infections in plants and animals

antimicrobial resistance

• Occurs when microorganisms acquire genes that help them survive antimicrobial stress

• Allows them to survive treatment

mechanisms of antimicrobial resistance

• Efflux pumps

• Decreased uptake

• Inactivating enzymes

• Target alterations

• Alternating enzyme

mobile genetic elements

• Segments of DNA that are capable of moving within genomes and/or among cells

plasmids

• Circular extrachromosomal elements

• Provide xenobolic/non-essential functions

  • Antibiotic resistance

  • Toxins

• Transmitted vertically through cell division

• Transferred between cells through transformation and conjugation

• Bacteria can have multiple copies

• Commonly used in cloning

plasmid incompatability

• Groups of plasmids that cannot stably coexist

• Plasmids from separate groups CAN COEXIST

• Incompatibility due to a shared ori or Par system

f plasmid

• A conjugative plasmid

• Original fertility plasmid

• Replication origin is utilized in BACs

mpf

• Mating pair formation

• Creates type IV secretion system

traa

• Subunit that forms the pilus of Mpf

• Pilus extension and retraction are ATPase-dependent

f plasmid transfer

• A single-strand nick is made in the F-plasmid on dsDNA

• SINGLE STRAND enters the recipient cell

• Complementary single-strand is synthesized in the donor

• Complementary single-strand synthesized in recipient

dtr

• DNA transfer and conjugation replication

  • oriT, TraI, M, Y

• Part of the F transfer apparatus

trai relaxase

• Nicks F plasmid at oriT

• 5’ is covalently attached to the Tyr residue of this

• Signaled by a coupling protein

• Makes a nick in the donor AND seals the nick in the recipient

  • Translocated to recipient cell with DNA

relaxosome

• TraI, TraY, TraM, IHF (host) system

hfr

• High-frequency recombination

• Method by which F can integrate into E. coli coli genome

• F can take portions of E. coli with it when it excises

• Can conjugate the entire E. coli coli genome

• Transfers genome COUNTER-CLOCKWISE

• Recombination frequency DECREASES with size

• Markers closer together have INCREASED recombination frequency

ices

• Integrative and conjugative elements

• Type of mobile genetic element that integrates into the host genome

  • Integrated on host/donor→ excises itself from the host genome

• Can be transferred between cells via conjugation

• Most common type of conjugative element

attachment sites

• ICES sites for integration into the genome

• NOT IS like for F-plasmid

int xis

• Integrase and excisionase

• Used for ICES integration into andexcision from the host genome during conjugation

helps nick

• Helicase processivity factor and relaxase

• ICES integration

ice induction

• ICE excision from the host and subsequent conjugation depend on the specific ICE

• Can result from:

  • SOS response

  • Cell-cell signaling

  • Stationary phase

  • Spontanaity

• Can cause host cell death or growth

griffith experiment

• S. pneumoniae is Gram+ pathogen

• Form smooth “S” colonies

• Dead pathogenic cells can transform cells into pathogens via transformation HGT

avery macleod maccarty experiment

• Concluded that transformation requires DNA without any cell-to-cell contact

  • Thus DNA is the genetic material of the cell

natural transformation

• Recipient cell has machinery to import external DNA

artificial transformation

• Treat cells with chemicals or electric fields to make them take up external DNA

• Occurs in a lab environment

• Ex. → Transformation with a plasmid

artifician transformation methods

• Chemically induced transformation

• Electroporation

chemical competence

• Treat cells with calcium ions, rubidium ions, etc.

• Add DNA

• Heat-shock cells

• Allow cells to recover with fresh medium and grow for a few doublings

• Plate and select for transformers

electroporation

• Wash cells to remove salts

• Add cells to DNA

• Electroporate

  • Temporarily permeabilize the cell membrane

  • Electric field brings DNA into the cell

• Allow cells to recover

• Plate and select for transformers

dna uptake

• Type II secretion system

• exernal dsDNA crosses external membrane of Gram- through PilQ

• dsDNA crosses cell wall via PilE/ComG

• ssDNA imported through cytoplasm via transmembrane channel

• ATPASE DEPENDENT

purpose of natural transformation

• Increase the generation of genetic diversity

• Obtain DNA to repair damaged genomic DNA

• Nutrient source via providing nucleotides for recipient cell

cloning

• Making multiple identical copies of a piece of DNA or genetically identical cells/organisms

• Same as plasmid/strain making

• Use natural transformation and HR to make new strains

amye

• Encodes amylase

  • Degrades starch

• Used to add constructs because can be disrupted without causing larger issues

ectopic

• The new location of a gene

endogenous

• The normal location where a gene is located

merodiploid

• Partially diploid

  • Contains its normal single chromosome complement plus a second copy of a limited segment of genetic material

• Ex.→ dnaG copies in endogenous locus and amyE locus in B. subtilis

complementation

• Restoration of a WT phenotype when two alleles are combined in the cell

  • Can be WT allele + mutant, or mutant + different mutant

• Intergenic, intragenic, or due to polarity

• Can be genomic or on a plasmid

• Cloning vectors can be used

bacteriophages

• Viruses that infect bacteria

• Contain capsids with genetic material, a lipid core, and tails

• Some filamentous

phage genomes

• linear dsDNA genome in the capsid

• Encode structural proteins, assembly genes, sometimes own replication and transcription genes

• Reliant on hijacking cell machinery and resources

lytic cycle

• Phage attaches and injects its chromosome

• Phage chromosome= replicated via the cell machinery

• Expression of phage genes to produce new structural components

• Phage assembly

• Release of progeny via lysis of the bacterial wall

host rnap expression

• Phage T4 can express genes using host RNAP

• Uses sigma 70 of the host to create g55

• g55 is used to express late genes→ capsid proteins and genome packaging proteins

t7

• E. coli lytic phage

• Encodes its own RNAP using bacterial machinery

• Uses RNAP to express late genes

• Highly specific promoter sequence

early middle genes

• Genes responsible for replication

late genes

• Genes responsible for packaging/structural components of new phage progeny

lysogenic cycle

• Phage infects host

• Phage genome is integrated into the host’s genome→ allows the phage to manipulate the host to create new progeny/grow

temperate phage

• A phage that can undergo the lysogenic cycle

• Also called a lysogenic phage

• Can carry virulence genes that convert host into a pathogen

prophage

• Lysogenic phage integrated into a bacterial genome

• Integration renders the host cell immune ot lysis by the same phage

lysogen

• A bacterium containing a prophage in its genome

attp

• Phage attachment site on host genome

• Used in prophage integration

attb

• Bacterial attachment site

• Specific DNA sequence on the bacterial host chromosome where a temperate bacteriophage integrates its genome during prophage formation

int

• Integrase in prophage integration

  • Mediates site-specific recombination

y recombinase recombination

• Cre recombinase recombines at loxP sites

  • Cre-lox system

• Y recombinase breaks one DNA strand at a time

• External E is required→ no ATP or GTP

• Strands are recombined at RBE sites

  • RBE= Recombinase binding element

s recombinase

• Breaks both DNA strands at the same time→ rotates strands to recombine

• Creates double-strand breaks and stays bound to DNA the whole time

• External E is required→ no ATP or GTP

• Cuts→ rotates→ reseals

moi

• Multiplicity of infection

• Average number of phage per bacterial cell

burst size

• Average number of progeny phage released after cell lysisb

phage crosses

• Infecting bacteria with two parental phages at a high MOI= production of a recombinant phage

• One bacterial cell can be infected with different phages simultaneously

• h= host-specificity

  • h+ can only infect one strain; h- can infect multiple

• r= rapid lysis/plaque size

  • r+ lyses slower/forms small plaques; r- lyses quickly/forms larger plaques

transduction

• The transfer of DNA from one cell to another cell via phage

generalized transduction

• Form of HGT in which a bacteriophage accidentally packages a random fragment of host bacterial DNA instead of viral DNA during the lytic cycle

• Phage is nonspecifc to DNA it takes up

• Phage cannot degrade bacterial DNA

cotransduction of multiple markers

• Closer markers= more efficient transduction

  • Recombination and phage head only fits so much DNA

• Markers far apart= reduced transduction frequency

• Used to map loci

specialized transduction

• Prophage does not excise properly→ takes nearby host DNA with it

• Mutant phage can lyse its host cell and infect a new cell

restriction modification systems

• Uses restriction endonucleases to target specific restriction sites on nucleic acids

• Methyltransferase methylates DNA at restriction sites to prevent degradation of its own DNA by restriction enzymes

• Non-specific targeting of non-host dsDNA

• Example of innate immunity

abortive infection

• Cell infected by phage can self-destruct before phage maturation

• Prevents phage infection at first compromization→ allows whole bacterial colony to survive

• Example of innate immunity

sgas

• Enzymes that promote abortive infection

crispr

• Cluster of short regularly interspaced palindromic repeats

• Mechanism of adaptive immunity in bacteria and archaea

• Type II is the most common type used in current genome engineering applications

cas genes

• CRISPR-associated genes are usually adjacent to the repeat locus

• Involved in the acquisition of elements or immunity

  • In Type II, Cas9 mediates cleavage of target DNA

tracrrna

• Hybridizes to the CRISPR repeat portion of pre-crRNA to mediate processing into mature crRNA

pam

• Protospacer adjacent motif

• First recognized by Cas9

• On the target, foreign DNA immediately adjacent to the protospacer sequence

prespacers

• Pieces of foreign DNA that will be incorporated into the CRISPRs

protospacer

• Targeting complexes bind to a complementary sequence in the target

• In the second phase of the CRISPR process/DNA targeting