micro exam 2

What is a NRPS? What do they produce?

complex enzyme that produces a peptide
Most antibiotics are produced by NRPS

DNA Structure

Double-stranded helix with antiparallel strands

DNA replication is ____

semiconservative
meaning each of the progeny strands have a parent and new strand with replication always going 5’—→ 3’

Rosalind Franklin

helped understand the molecular structure of DNA

DNA polymerase

catalyze addition of dNTPs
DNA polymerase III is largely involved in DNA replication

Primase

lays RNA primer to start replication

How does DNA polymerization work?

3’ OH attacks the 5’ phosphate and forms a phosphodiester bond
must have 3’ OH for polymerization to work

Replication Fork

zone of unwound DNA where replication occurs

helicase

unwinds DNA

DNA extension occurs continuously on the ____ strand and discontinously on the ____ strand, known as Okazaki Fragments

1. leading

   1. lagging

replisome

complex of multiple proteins involved in replication

exonuclease

removes RNA primer

DNA ligase

seals nicks in DNA

Plasmids

small, circular, double-stranded DNA molecules that carry nonessential, but advantageous, genes
typically carry antibiotic resistance

why use 16S for bacteria identification?

it has highly conserved regions and variable regions which makes it ideal for identification

PCR

amplifies specific DNA regions

kary mullins

invented PCR

PCR applications

phylogenetic studies, microbe identification, forensics

Agarose gel electrophoresis

visualize and separate DNA and PCR products by using an electric current to move charged DNA through the gel matrix
small products migrate faster than large

Sanger Sequencing purpose

determine the order of nucleotides in a segment

Sanger Sequencing Process

ddNTPs and dNTPs used together
ddNTPs are labeled with a fluorophore
ddNTPs stop the sequence

PacBio or SMRT Sequencing

Uses small sequencing chambers and phospho-linked nucleotides
when nucleotides are added, the fluorophore is detected by the computer

Oxford Nanopore Technology

pass single strand of DNA through a protein pore
does not use dNTPs

PacBio and Nanopore vs. Sanger

PacBio and Nanopore are better for whole genome sequencing fast and cheaper
Sanger is only one that can be used for PCR products

ORF

encodes a protein

Bioinformatics

combines biology, computer science, and statistics to predict gene functions including metabolic pathways, antibiotic resistance, and biosynthetic clusters

What produces genetic diversity in bacteria?

horizontal gene transfer

conjugation

plasmid transfer

transduction

transfer by viral delivery

transformation

transfer of free DNA

Vertical Gene Transfer

gene replication and cell division

3 types of genes to determine a species

core: genes shared by all strains
flexible: variable genes present in some strains
pan: includes all core and variable genes

Taxonomy

science in classifying organisms

Bacterial species concept

A group of strains that frequently exchange, or could exchange, genes and are relatively restricted from such exchange with other groups. As a result, a species shares core genome content and DNA similarity, compared to other strains.

Phylogenetics

study of evolutionary history among individuals

Polyphasic approach

phylogenetic analysis - relatedness based on gene sequences
genomic analysis - compare genomes
phenotypic analysis - identify biochemical characteristics

Carl Woese

identified 16S gene can be used for phylogenetic studies
established 3 domains of life: bacteria, archaea, and eukarya

How did we create our phylogenetic tree?

PCR the 16S rRNA sequence, then go through Sanger sequencing, BLAST to identify genus, align sequences

Multi-locus sequence alignment

sequencing of housekeeping genes to make a phylogenetic tree
MLSA distinguishes between closely related strains

DNA-DNA hybridization

known species’ genome is sheared, denatured, and labeled with P32
sheared and unlabeled DNA from known and unknown organisms are bound to nylon membrane
measure radioactivity to compare similarity

What indicates similarity between strains in DNA-DNA hybridization?

70% or higher hybridization values

Average nucleotide identity

compare 2 genomes and if ANI is >96%, it suggests similarity

LUCA

occured in thermal vents
1st cellular life
4.0 BYA

Formation of earth

4.5 BYA

Bacteria and archaea divergence

3.7 BYA
differ in lipid chemistry, transcriptional and translational machinery, and DNA organization

Cyanobacteria evolved and O₂ is generated

2.7 BYA

endosymbiosis

2.0 BYA
Theory that eukaryotic cells arose from prokaryotes

eukaryotic cells arose from___

H producing bacteria and archaea, which became mitochondria, and acquired a nucleus
explains why lipids, translational machinery, and transcriptional machinery are similar in archaea and eukarya

plants evolved from___

cyanobacteria which formed chloroplasts

first eukaryotes

1.7 BYA

multicellular eukaryotes

1 BYA

support for endosymbiotic hypothesis

mitochondrial genome encodes rRNA, tRNA, and proteins
16S rRNA gene is similar to rickettsia, which produces ATP like mitochondria
Chloroplast genome encodes proteins for photosynthesis

Rickettsia

genus of nonmotile, gram-negative, nonspore-forming, and obligate intracellular bacteria
pathogenic species are transmitted by ticks and cause typhus, african tick-bite fever, and rocky mountain fever

how do you treat diseases caused by rickettsia?

tetracycline

how does lactose enter the cell?

transported by lac permease symporter which is regulated by the lac operon

operon

genes organized in same direction and co-transcribed

what is lactose?

disaccharide made of glucose and galactose

Phosphotransferase System

transports glucose, fructose, and mannose into the cell
increase in Enzyme II phosphorylation activates adenylate cyclase, which converts ATP to cAMP which binds CAP to regulate lac

lac Z

Encodes beta-galactosidase
cleaves lactose to become galactose and glucose
also converts lactose to allolactose

lac y

permease

lac A

acetyltransferase

lac I

binds to operator which prevents RNA polymerase from binding to promoter and expressing lac

what happens when cAMP binds CAP?

stronger RNA polymerase binding to promoter

allolactose

binds lac I and removes it from operator

no lactose

low lac operon expression
lacI is bound to promoter

lactose present

intermediate lac operon expression
RNA polymerase bound to promoter

lactose present and glucose low

High lac operon expression
cAMP bound to CAP and RNA polymerase bound to the promoter

4 characteristics of active transporters and how it applies to lac operon

specific - lac operon only aids in lactose transport

regulation - lactose and glucose presence influence expression

energy - gets energy from proton motive force

saturation - has a maximum amount of lactose it can bring in

low glucose means what?

high cAMP and phosphorylated enzymes

what facilitates ATP —→ cAMP

adenylate cyclase

NCBI

national center for biotechnology information

BLAST

basic local alignment search tool

septum

inward growth of the cell and the location of cell division

FtsZ

forms ring in center of the cell to control where cell divides

ZipA

anchors FtsZ to cytoplasmic membrane

FtsA

anchors FtsZ and recruits cell division proteins

FtsI

transpeptidase

FtsK

assists in chromosome separation

What ensures FtsZ ring forms properly?

Min proteins which oscillate between poles to block polymerization

MinD-ATP

recruits MinC
binds membrane

MinCD

inhibits FtsZ polymerization

MinE

displaces MinCD by ATP hydrolysis

What is GFP?

Green Fluorescent Protein
used in research as a reporter gene to visualize proteins

What was GFP first purified from?

Aequorea victoria

autolysins

cleave existing peptidoglycan

bactoprenol

flips peptidoglycan units outside of the cell for peptidoglycan synthesis

transglycosylase

enzyme that covalently bonds sugars in peptidoglycan units

transpeptidase

enzyme that covalently bonds peptides in peptidoglycan units
inhibited by beta-lactams

MreB

shape determining protein in prokaryotes
makes spiral bands which form bacillus-shaped bacteria

transpeptidation

cross-links D-Ala and DAP

What antibiotics inhibit transpeptidation?

penicillin, cephalosporin, carbapenem, vancomycin

What is labeled in Sanger Sequencing?

ddNTPS labeled at their 3’ end

PacBio SMRT vs. Oxford Nanopore

• PacBio uses a fluorescent dNTP while Nanopore uses ionic current changes

• PacBio is best at HiFi reads with structural variants and Nanopore is best at very long genes

What is labeled for PacBio SMRT?

dNTPs labeled at their phosphate group

Interpreting BLAST results

• Percent Identity → >90% indicates close homology

• Query Coverage → >80% indicates most of your sequence matches hit

• E-Value → 0 indicates it does not match other sequences in database