[BIO 120.11] Module 3 Part 4: Enrichment to Quorum Sensing

TRUE

T/F: Quorum sensing systems are found in both bacteria and archaea

FALSE

T/F: Quorum sensing systems are only found in bacteria

behaviors, cell density

A quorum sensing system is a communication system that regulates ___ that require a certain ___ to be successful

Sufficient Numbers

Quorum means?

Cell density dependent regulation of bacterial gene expression

What is the precise definition of quorum sensing?

Signal threshold

What is the real basis for determining whether a culture will exhibit individual or group behavior?

Autoinducers

Signals that switch on QS

Nonpeptide, organic molecules

What are the autoinducers in Gram negatives?

Small peptides

What are the autoinducers in Gram positives?

Small peptides

What are the autoinducers in Archaea?

TRUE

T/F: Signal diffuses freely across the cell envelope in either direction

FALSE

T/F: Signal diffuses freely across the cell wall in either direction

FALSE

T/F: Signal diffuses freely across the cell envelope towards one direction

Acyl-homoserine lactones (AHL)

What is the signal for proteobacteria?

LuxI

What is the signal synthase for Proteobacteria’s AHL?

Signal synthase for AHL

What is LuxI in Proteobacteria?

LuxR

What is the transcription regulator in proteobacteria?

Transcription regulator

LuxR acts as a __ among proteobacteria’s AHL QS systems

FALSE

T/F: The active form of the LuxI protein is a dimerized form

FALSE

T/F: The active form of a LuxR protein is a monomer

TRUE

T/F: The active form of a LuxR protein is a dimerized form

Lux Region

Region within LuxR wherein AHL signals bind?

Bioluminscence

A very common phenotype positively regulated by QS

FALSE

T/F: Bioluminescence is a very common phenotype negatively regulated by QS

TRUE

T/F: Bioluminescence is a very common phenotype positively regulated by QS

Aliivibrio fischeri

Light organ symbiont

Euprymna scolopes

Allivibrio fischeri is a symbiont of what species?

Antibacterial activity against nosocomial pathogens

What is a benefit of isolating bioluminescent Vibrio from fishes?

S. aureus, K. pneumoniae

What are 2 nosocomial pathogens that have been studied against isolated Vibrio species?

S-adenosyl methionine to AHL

What conversion reaction does AHL synthase catalyze?

Promoter region

The light green region in the image is ___

Upregulation of lux operon transcription

What is the net effect of binding of AHL to luxR in lux operon?

Lux operon

Operon responsible for Vibrio bioluminiscence

Biofilm, Swarming, Virulence, Pyocyanin

4 QS-Mediated Bacterial Cooperation P. aeruginosa? Bio Swarm VP

LasR, IqsR, RhlR, PqsR

What are 4 QS systems in P. aeruginosa?

LasR

What is the most powerful QS system in P. aeruginosa as it controls the expression of other QS systems?

Secreted toxin (cooperators are resistant, cheaters are susceptible)

How are social cheaters selectively killed?

Toxin resistance factor

What is coded for by the cooperator QS systems that allow it to survive toxins that are supposed to selectively kill social cheaters?

Hydrogen cyanide, Rhamnolipid, Pyocyanin

What are 3 examples of public goods for P. aeruginosa?

Their usage of freely available public goods can cause a population collapse

How do social cheaters “exploit” cooperators?

Shiga Toxin

What virulence factor in E. coli is controlled by QS?

AI-3

What are the signals for Shiga toxin production in E. coli?

More E. coli means more AI-3 signals that can induce Shiga toxin production

How does having more E. coli increase its pathogenicity?

Arginine operon

What operon in S. aureus influences virulence factor production in S. aureus?

AIP or autoinducing peptide

What is the signal for virulence protein production in S. aureus?

AI-3 synthase produces AI-3 → AI-3 and Intestinal hormones activate sensor kinases → Sensor kinases activate transcriptional regulators → Transcriptional regulators activate enterotoxin production

What is the flow of Shiga toxin production in E. coli?

Basal transcription of Arginine operon produces pre-AIP → Pre-AIP is converted to AIP by ArgB → AIP exported out of cell → AIP binds to ArgC causing autophosphorylation → ArgC autophosphorylates ArgA → ArgA-P activates expression of genes required for pre-AIP and virulence proteins

What is the flow of virulence protein production in S. aureus?

Present in small numbers, Grow slowly

Enrichment techniques are employed for bacteria with the following 2 physiology:

Oil degrading bacteria

What is an example of bacteria that must have an enrichment technique before isolation?

Optimal nutrient and physical condition

Enrichment techniques involve a combination of?

Colony morphology, Cell morphology, Gram reaction

What are 3 Methods to verify purity of culture?

Colony

Visible, isolated masses of cells

FALSE

T/F: Colonies are indicator of composition and actual purity of culture

TRUE

T/F: Colony morphology is an indicator of composition and presumptive purity of the culture

FALSE

T/F: The colony morphology alone is already enough for establishing purity

TRUE

T/F: The colony morphology is a good indicator for determining if it is okay to proceed to Gram staining

Appearance and uniformity of colonies on a Petri plate

What is the criteria for culture purity?

Culture condition, Nutrient supply, Other physiological parameters

What 3 parameters can influence shape, color, and size of colonies?

TRUE

T/F: It is possible to have mixed Gram - and + cells in a pure colony

FALSE

T/F: If you have a mix of Gram - and + cells, we can automatically declare it as nonpure

Retain viability, Maintain purity, Maintain trait of being “true-to-type”

What are the 3 goals of culture preservation? ViPiTTT

Periodic transfer to fresh media, Overlaying with mineral oil, Freeze Drying or Lyophilization, Freezing in liquid nitrogen, -80 ultralow freezer

What are the 5 culture preservation methods? POL LiNi UF

Time of interval of transfer, Culture medium, Storage temperature

What are 3 factors to be considered for periodic transfer to fresh media?

Limits the availability of O2 and reduces the metabolic rate

What is the principle behind overlaying with mineral oil or glycerol?

TRUE

T/F: The viability of colonies in overlaying with mineral oil or glycerol varies with species

FALSE

T/F: The viability of colonies in overlaying with mineral oil or glycerol is consistent among all species

Inoculum

Stocks preserved in mineral oil or glycerol can serve as a source of ___ while preserving the initial culture

Freeze drying or Lyophilization

Culture preservation method that employs rapid drying in a frozen state

Fast freezing

Freeze drying or lyophilization is also known as?

Long term survival, Low mutation rate, Small storage container

What are 3 advantages of freeze drying?

Ampoules of powdered bacteria

What is the product in freeze drying?

-196 deg C

What is the temperature involved in freezing with liquid nitrogen?

Freezing with liquid nitrogen

Specimens are frozen along with a protective agent (glycerol) in liquid nitrogen

Glycerol

What is a protective agent commonly used in freezing preservation techniques?

-80 to -86 deg C

What is the temperature involved in ultralow freezing?

Ultralow freezing

Specimens are frozen along with a protective agent (glycerol)

Phenotypic Analysis, 16S rRNA gene sequencing, Genomic analysis

What are 3 types of bacterial identification techniques?

Phenotypic analysis

Bacterial identification technique that involves observable characteristics that can be used to differentiate species

FALSE

T/F: In phenotypic analysis, you only need to describe your observations of your sample

TRUE

T/F: In phenotypic analysis, there is a need for a point of comparison to prevent mere characterizationP

Phenotypes are growth-condition dependent, so morphologies in natural habitats versus lab cultures may be different

What is a caveat of phenotypic analysis?

Dichotomous Key

Method of determining species based on a series of phenotypic characteristcis

Bergey’s Manual of Systematics of Archaea and Bacteria, The Prokaryotes

What are 2 References for Bacterial Classification System

FALSE

T/F: The reliability of phenotypes for systematic analysis is consistent across all taxonomic groups

TRUE

T/F: The reliability of phenotypes for systematic analysis is varied with taxonomic groups

API Strips

Testing Kit composed of 20 miniature biochemical tests with a software

DNA Extraction → PCR → DNA Library Construction → DNA Sequencing → Data Analysis

What is the workflow in a 16S rRNA gene sequencing process?

16s rRNA

Highly conserved region among all prokaryotes

V1-V9 regions

Regions of variability across prokaryotes

FALSE

T/F: The 16S rRNA is a highly conserved region among bacteria

TRUE

T/F: The 16S rRNA is a highly conserved region among prokaryotes

96% for ANI since this is relative to genome, 98% for 16S rRNA gene sequence similarity

What is the % similarity expected for ANI and 16S rRNA gene sequence similarity before we can declare that we have same species?

< 97% similarity

What similarity % is required before we can declare that two species are possibly new?

Genomic analysis

Use of entire genomes for the identification and description of bacteria

Library Construction → Random sequencing phase → Closure phase → Complete genome sequence

What is the workflow for genomic analysis?

Comparative analysis of gene content, Synteny, G + C content

What are 3 types of genomic analysis?

Synteny

gene order in the genome

15,000 sequences per Mb

How many sequences per Mb is involved in the random sequencing phase of genomic analysis?