microbiology test 3

prokaryotic nucleoid

contains the genome which consists of a single circular DNA molecule that encodes all of the essential genes required for bacterial growth

prokaryotic plasmid

small, circular piece of DNA that has non-essential genes

dna replication

bacterial genome is copied following DNA replication

origin of replication

is the specific location on the DNA molecule where DNA replication begins, allowing the DNA to be unwound and copied.

bacterial structure

single circular chromosome but some linear, one origin of replication

archaea structure

single circular chromosomes, multiple origins of replication

bacteria packaging

primarily negative supercoiling but aided by DNA gyrase to induce supercoiling

archaea packaging

use of negative supercoiling and histones

archaea thermophile packaging

use positive supercoiling due to more stable structure

bacterial proteins

uses nucleoid associated proteins to help for scaffolding supercoiled DNA

archaea proteins

use both histones to form nucleosomes and nucleoid associated proteins

prokaryotic initiation

RNA polymerase and sigma or transcription factors associate with the promoter site to form an active RNA polymerase complex

prokaryotic elongation

DNA strands are separated and RNA is synthesized

prokaryotic termination

RNA polymerase complex reaches the termination site at the end of a coding region and releases from the DNA template

bacteria promoter

use -10 pribnow box and -35 recognition sites which are recognized by sigma factors for RNA polymerase complex assembly

-10 pribnow box

number of nucleotides before the nucleotide being transcribed

archaea and eukaryotic promoter

TATA box and a B recognition element site for transcription factors to bind

sigma factors

help RNA polymerase bind the correct promoter and activate it

RpoD sigma factor

most common and regulates “housekeeping” genes

operon

transcriptional unit comprised of multiple adjacent genes that are under the control of a single promoter

trp operon

encodes machinery to produce tryptophan

lac operon

encodes machinery to use lactose, negative inducible, turned on by an inducer

how are operons turned on or off

through the binding of regulatory proteins near or on the promoter

regulatory genes are

constitutively expressed which means they are always on

operator

the binding site on the operon which is used for gene regulation

repressor

shuts off the operon

activator

turns on the operon

if low lactose

dont need lactose machinery, dont activate the lac operon, repressor is active

if high lactose

need lactose machinery, activate lac operon, repressor is inactive, inducer is present

repressor is controlled by binding of

an allolactose

allolactose

an inducer, molecule which inactivates a repressor

3 lac operons that encode 3 genes which produce 3 enzymes

LacZ, LacY, LacA

LacZ operon

encodes a beta-galactosidase enzyme which is responsible for cleaving lactose into glucose and galactose

LacY operon

encodes a galactoside permease enzyme which is embedded in the cell membrane and transports lactose into the cell

LacA operon

encodes a transacetylase enzyme which acetylates glucosides, galactosides, and lactosides

glucose

primary sugar energy source for most prokaryotes and eukaryotes

diauxic growth

2 different sources are available for metabolism, microbe will use the source that is easier to metabolize first

log phase

associated with consumption of the first sugar

lag phase

cell activates the operon necessary to utilize the second sugar

negative control

regulator protein is a repressor

positive control

regulator protein is an activator

inducible

expression is off until turned on

repressible

expression is on until turned off

4 possible combinations

inducible negative control, inducible positive control, repressible negative control, repressible positive control

inducible negative control

repressor protein normally blocks transcription, but the presence of an inducer molecule inactivates the repressor, allowing gene expression to “turn on”

inducible positive control

gene expression is turned on only when a specific activator protein is present and bound to the promoter

repressible negative control

an operon is expressed by default but is turned off when a specific signal molecule binds to a repressor protein, enabling it to block transcription

repressible positive control

transcription is normally ON because an activator protein is active, but transcription is turned OFF when a repressor molecule inactivates that activator.

auxotrophs

organisms that have experienced a mutation that makes a nutrient acquisition process dysfunctional

ames test

use of auxotrophs in the presence of test reagents to evaluate the strength of potential mutagens

plasmids

small, circular dna molecules that are often responsible for rapid horizontal gene transfer between prokaryotes

pathogenic factors

genes associated with increases disease or pathogenesis, and r factors

horizontal gene transfer

transfer between two individuals within the same generation

vertical gene transfer

transfer through replication to subsequent generations

expression

genetic information is used within a cell to produce the proteins needed for the cell to function

recombination

genetic information can be transferred horizontally between cells of the same generation

replication

genetic information can be transferred vertically to the next generation of cells

4 main mechanisms that bacteria use to acquire new DNA

mutagenesis, transformation, conjugation, transduction

mutagenesis

mutations may alter genes making organisms acquire new traits

transformation

uptake of free DNA directly from their environment and incorporation into the gene by recombination

conjugation

transfer of DNA from on bacterium to another via a sex pilus, incorporation in the genome is achieved by recombination

transduction

transfer of DNA via bacteriophages followed by recombination

mutations can occur by the following mechanisms:

errors in DNA replication, spontaneous chemical modifications, exposure to mutagens

susceptibility to DNA Damage

prokaryotes are more susceptible to mutagenic events due to no space in genomes

higher evolutionary rate aided by selective pressures

due to high rates of replication of prokaryotes, favorable mutations are rapidly selected for in populations

base substitutions due to errors in the DNA polymerase

if the mutation is not caught the mutations could become permanent

slippage during replication causing frameshifts

looping and hairpins can result in mismatch and insertion or deletion of nucleotides

positive (direct) selection

detects mutant cells because they grow or appear different

negative (indirect) selection

detects mutant cells that cannot grow or preform a certain function

replica plating

used to transfer the same colonies to different plates to evaluate sensitivities

frederick griffith

transformation of genetic material, identifying the role of DNA in heredity

s strain

virulent (disease causing), polysaccharide capsule that protects it from the immune system

R strain

non-virulent (harmless), lacking a capsule

steps of transformation

DNA release, DNA uptake, integration, gene expression

DNA release

bacteria die or break apart and release DNA fragments into the environment

DNA uptake

a living bacterium takes up the free DNA

integration

the foreign DNA can integrate into the hosts genome by recombination

gene expression

the cell expresses new genes from the acquired DNA

ways to increase transformation efficiency

salt treatment, electroporation, heat shock

salt treatment

makes membranes more permeable

electroporation

electric pulse temporarily opens pores

heat shock

thermal imbalance causes membrane leakiness

f plasmid

replicates once per cell cycle and segregates into the daughter cell

transfer gene region (tra)

encodes the machinery associated with F pilus formation to promote conjugation between cells

OriT

origin for transfer for copying the plasmid during cell transfer

IS2/3 and TN100

insertion elements or simple transposons which copy and insert themselves randomly

high frequency recombination cells (HFR)

cells with the F plasmid fully integrated

rolling circle replication

chromosomal genes transferred will be transmitted to the F- cells in one direction of origin

prophage

integration of the phage genome

lysogen

bacterial chromosome of a bacteria

generalized transduction

phage infects bacterial cell, phase DNA and protein are made, bacterial DNA fragment is packaged in a phage capsid, crossing over, recombinant bacteria

specialized transduction

bacterial cell has prophage integrated between gene A and B, prophage DNA exits incorrectly, phage particles carry bacterial DNA and phage DNA, crossing over, recombinant bacteria

restriction modification system

defense mechanism established in bacteria and archaea to destroy foreign DNA that enters the cell

restriction endonuclease

recognizes and cuts specific nucleotide sequences found within foreign DNA, named based on species, specific strain, and number of restriction enzymes isolated

methylase

attaches S-adenosylmethionine (SAM) to restriction site sequences to protect suites from being cut

three main types of restriction modification systems

Type 1, Type 2, Type 3

Type 1 RMS

a single protein which encodes both methylation and endonuclease domains, distant recognition

type 2 RMS

separate methylase and endonuclease enzymes, at or close recognition site

type 3 RMS

single protein which encodes both methylation and endonuclease, but recognition is close to cleavage site

molecular cloning

using bacteria to copy foreign DNA of interest