chapter 9, sensing and responding to the environment

some features of an environment that a microbe would be responsive to

nutrient availability: needed for growth, E, reproduction. upregulate nutrient transporters and express high affinity

osmotic pressure: high salt/ low water= shrinkage plasmolysis. low salt/ high water= bursting lysis

presence of toxins/ antimicrobials: toxins can damage DNA in microbes, proteins, or membranes which can lead to cell death. Microbes form biofilms and do HGT

signal: chemorepellents

cells move away from high [chemicals] gradient

signal: chemoarrtactants

cells move towards high [chemicals] gradient

response

changes in gene expression & phwnotype

signal transduction

• process where microbes detect external signals from the outside environment and convert the signals into a cellular response within the cell. (External signals: chemical gradients, pH, osmolarity, light, magnetic fields, nutrients, toxins.)

• Microbes sense and respond!

• This is an immediate response.

• 1 or 2 component systems

• to respond to stimuli, sometimes proteins do NOT need to be made, sometimes they DO need to be made

Protein- based signal transduction systems:

they are needed to sense and respond to the changes in the environment

sensor kinase

• protein that detects a signal

• always embedded in the membrane

• It phosphorylates the response regulator. (Phosphorylates: process of adding a phosphate group (PO4^3-) to a protein or an other molecule/ amino acids)

• It is ATP- dependent, from ATP → ADP it takes a PO4^3- from ATP to phosphorylate. This consumes a lot of E

response regulator

• protein that initiates change (gene expression, enzymatic activity, and protein binding)

• membrane OR cytoplasm

• ONLY activated by phosphorylation to exert its activity

• it undergoes a conformational change that can actively bind to the DNA P

one component system

• more likely to be found in prokaryotes than two component systems

• 1 protein, 2 domains

• A single protein acts as both sensor and response regulator. with 2 functions

• regulator is attached to sensor, can be an activator or inhibitor

• DNA P needs to go to the system. cell needs to reorganize the inner cell

two component systems

• 2 separate proteins/ domains, sensor kinase domain is membrane bound, response regulator is in the cytoplasm

• response regulator can go to the DNA P instead of cell inner organization

• this leads to a faster response

output domain/ effector domain

DONT NEED TO KNOW Yyayyayayya

• part of a response regulator protein that executes the cellular response after RR is phosphorylated.

• It determines the action the protein takes

• The responses for the systems are dependent on the function of the output domain

response regulators (RR)

use output domains to execute cellular responses after phosphorylation. There are 3 broad functional classes:

1. Enzymatic activity

2. Bind to other proteins

3. Bind to DNA to affect transcription/ gene expression (activate/ repress)

   1. Majority of output domains in prokaryotes bind to DNA

   2. Their primary role is to affect transcription & the synthesis of new proteins, this overall mediates the cellular response

plasmids

• extra- chromosomal DNA that is NOT part of the genome/ main chromosome the genes that are encoded are still part of the genome even though they are not actually in the chromosome.

• They are circular and self- replicating.

• They encode non-essential genes.

• They can be shared by HGT  (conjugation: when 2 prokaryotic cells transfer a plasmid when they directly touch conjugative plasmids via their pilli).

Laboratory plasmids (general)

• are genetically engineered and introduced into cells by transformation → transform the plasmid into a recipient cell (chemically/ elector competent) (transformation: transport of naked DNA from a lysed cell into a recipient. The uptake of free environmental DNA by a cell)

• engineered circular DNA used for cloning, expression or gene editing

Laboratory plasmids (features)

1. Origin of replication (Ori): where plasmid replication starts. It can replicate independently of the main chromosome

2.  Multiple cloning site: where you insert DNA (ex// gene of interest). This area of plasmid has nothing important, so a gene can be inserted. “Multiple cloning stite”, can use endonucleases/ enzymes that cleave the DNA too insert genes. This allows the target gene to be cloned and amplified to produce proteins of the gene.

3. Selectable marker(s): how you select for bacteria carrying plasmid and let them grow; if the gene is not in the plasmid it will die. It llows bacteris to grow when plasmid is taken up inside. You dont want cells that are not carrying a plasmid to take up the resources. They are genes included in the plasmid so scientists can identify and grow only the bacteria that successfully took up the plasmid. 

   1. Not all microbes will take up the plasmid. Transformation efficiemcies arw ~75%. You dont wabt other 25% to replicate, gene of interest is not there

   2. Antibiotic resistance gene: genes that allow bacteria to survive in the presence of antibiotics. They encode an enzyme that either inactivates the antibiotic or pumps the antibiotic out of the cell

      1.  You can grow microbes that have taken up the plasmid in the presence of the antibiotic while the others die

Alternative selectable marker: allow bacteria to metabolize a nutrient of form a pigment. Artificial sugars that dye bacteria

steps to genetic engineering

1. Design your interested plasmid. Use parts of the gene you want to study!

2. Transform your cells. Make small pores in the cell membrane so plasmid can slip in

• Low efficiency: many cells die and/or do not uptake a plasmid

3. Selectively expand the transformed cells

• Grow the bacteria in selective conditions (ex: on agar containing antibiotic)

• Cells that don’t have the plasmid (with selection marker ex: antibiotic resistance genes) cannot survive

4. Success! The bacteria that grow have your plasmid

• How can you confirm the bacteria actually contain the plasmid?

lab plasmids: expression proteins

• know: promoter

• unknown: gene

• determine: coding and function of the gene

• multiple cloning sites in front of existing P

• can grow bacteria and enforce expression of the protein of interest

• clone plasmid downstream of P

• Bacterial in nature: share features typical to bacterial proteins

lab plasmids: reporter w

• know: gene (GFP, beta gal, DETECTABLE PROTEIN) (Green fluorescence protein(GFP): one common reporter protein, Fluorescence levels are measured. From jellyfish. Indicates that the promoter is either being actively transcribed or not) (lacZ (beta-galactosidase) enzyme: 2nd common reporter protein, beta-galactosidase activity levels are measured. Indicates that the promoter is either being actively transcribed or not)

• unknown: promoter

• determine: if a gene of interest is being expressed. under what conditions does the P work under. the promoter can tell us when/ in what qualities the reporter protein is expresser protein is expressed. determine condition that activate expression of the target gene. to study how a gene is regulated

• promoter of the gene is cloned in front of a reporter protein

• insert only the P of gene of interest into reporter plasmid

novel genes

• promoter + operator + gene

• a newly discovered/ previously uncharacterized gene that has not been documented

• investigate by Deleting the gene from the DNA to see what cells can/ cant do without it. This figures out exactly what the gene does specifically

knock-out/ deletion mutants

• bacterial strains made in lab where researchers purposely deleted a gene of interest from its chromosome and see what happens to the phenotype of the studied gene

• can be done for both expression and reporter plasmids

• problem: dont know if changed phenotype is from polar effects (polar effects = side affects, great effects of growth, regulation, expression of other genes)

complementation

• compliment the mutant when a gene is deleted to confirm the change caused was NOT caused by polar effects/ side effects/ mutation.

• They do this by re-introducing the original gene on an expression plasmid into the cell; this should revert the phenotype to normal. This gene can have its own promoter. (Expression plasmids: produce a protein of interest.)

• If gene phenotype did NOT return to normal the change was due to polar effects

• A compliment is a control condition makes sure the effects are from the gene and ONLY from the deletion of the gene, not a mutant