MIC 102 Exam 3

List 2 examples of multicellular/cooperative behavior from Myxococcus xanthus:

1. Predation- feed/hunt on other microbes in soil 2. Development-fruiting body formation

List the basic steps of biofilm formation (3):

1. attachment (reversible) 2. growth (inside an excreted EPS matrix) 3. detachment Motile when on their own and non-motile when they join the biofilm.

Heterocysts are anoxic, what 2 things are you increasing to accomplish this?

1. increase # cell wall layers 2. increase glycolipid composition

Myxo uses _ to regulate many things including the process of development.

2 component systems

_ are a form used in cyanobacteria when they're starved or under temperature stress. They have added layers of cell wall and they are resting because most of their metabolism is shut down.

Akinetes

What is an example of a biofilm in humans?

Plaque formation on dentition (other random examples-piping

A mixture of peptides produced by protease activity on myxo cell surface proteins. Acts as a quorum sensing mechanism driving aggregation. Name the signal!

Signal A

If you make a mutation in one of the extracellular signals for fruiting body formation what happens?

You can block development.

Control of C. crescentus differentiation is similar to ?

concept of sporulation

Myxobacteria are diverse but tend to be _ in structure.

simple

When DnaK runs out of proteins to fold it will take _ bringing it to a FtsH protease to degrade it. So when no more heat stress we can turn off this heat shock response.

σH

Be able to compare endospores & exospores:

...

P. aeruginosa (soil bacteria/can be opportunistic pathogen in immunocompromised humans) biofilm formation is controlled by quorum sensing systems. Describe these 2 systems:

1. 1st quorum sensing system (LasR+AHSL=3-oxo-C12-HSL production) that helps to set up the biofilm (are there enough of the bacteria to attach to surface and grow out & uses one type of gene system). This is the control system 2. 2nd quorum sensing system (RhIR+AHSL=C4-HSL production) is once the cell density reaches a high enough stage it kicks off this system. 2 quorum sensing systems use 2 different autoinducers & 2 sets of luxR- luxI homologs (lasR-lasI and rhlR-rhlI)

2 different motility systems controlled by 2 different genetic systems, list the motility systems:

1. Adventurous (a-motility)- a form of gliding motility where the cells move independently (hypothesis of movement-slime nozzle propulsion and/or focal adhesion "tank tread" movement-red/green in pic) 2. Social (s-motility)- twitching with type IV pilus, requires LPS, & cells move together as swarms (attached together with pili as well)

The dental plaque biofilm has many different bacterial species involved, ecological succession happens as the biofilm is growing. New types of bacteria are recruited at different stages, list the steps (3):

1. Association and Adhesion of individual bacteria (Mostly G+ cocci like Streptococcus mutans) 2. Proliferation and development of microcolonies of these bacteria (New species added all the time, some are attracted to signals produced by existing members of the biofilm) 3. Biofilm formation and maturation

Throughout the morphological phases there are 5 extracellular signals being passed between cells to coordination fruiting body formation. List the 5 signals: (said don't need to memorize specifics of the signals)

1. E-who cares, function unknown? 2. B signal- active at 0hrs, and turns a couple of genes on to push us through development, first signal 3. A signal- active around 2hrs 4. D signal- active 4hrs 5. C signal- active around 8hrs.

List some characteristics of Myxococcus xanthus (5):

1. Gram- soil bacteria 2. Obligate aerobe, metabolism based on aas 3. Widespread (found in any soil, also marine ones) 4. Non-pathogenic 5. Lives cooperatively (biofilm or biofilm-like but motile)

Cells in the fruiting body differentiate to different forms, list 3 fates of vegetative cells during development:

1. Myxospore-in the middle (not endospore, similar to akinete in cyanobacteria) 2. Peripheral rod- make up the structure of the fruiting body 3. Autolysis- kill themselves to provide their nutrients to the rest of the developing cells in the fruiting body (similar to Bacillus sporulation) 4th fate! Don't make it into fruiting body and wander off

List the 2 cell types of Caulobacter crescentus:

1. Swarmer- flagellated, piliated, motile, nonreproductive. (replication machinery shut off) 2. Stalked- non-motile, reproductive (produce swarmer cells, unequal division). lack a pili & flagella and end up with a stalk. Form of differentiation, related to nutrient levels but not really a stress response, is apart of the life cycle

List things that can happen in response to a stress:

1. biofilm 2. heat shock response

There are many ways to get to stationary phase in a culture, list them (5):

1. carbon source depletion 2. oxidative stress 3. envelope damage 4. osmotic stress 5. low temperature Any one stress or combination of stresses (if strong enough) can lead to the same general outcome of deciding to go into the stationary phase. common to have a buildup of all of the stresses.

Development is a highly regulated process to get from starvation to fruiting & sporulation. The main idea is the myxo cells need to sense that they're starving & need to communicate that they're starving with other cells. List the 2 branches:

1. cellular starvation branch of the response 2. population starvation branch of the response.

What genes will be turned on in stationary phase (7)?

1. genes to make more flagella & pili (to help get out of stationary phase) 2. change enzyme activities for more recycling and biosynthesis 3. LPS is upregulated 4. membrane structure composition 5. PG is simplified and stronger with more cross links 6. nucleoid condensed (no replication soon) 7. cytoplasm composition changes

List the 3 2-component systems of myxo:

1. normal (histidine kinase & response regulator) 2. orphan (histidine kinase or response regulator) 3. hybrid (histidine kinase/response regulator)

Two component systems are often key components in stress responses. Response is typically carried out by (3):

1. protein sensor (does not always have to be a protein) 2. a transmitted signal that affects gene expression 3. feedback control mechanism (to turn it off)

Responses to stress tend to follow a pattern, list the general steps after a stress is applied (3):

1. sense the stressor (starving at this point & need to fix it) 2. respond to stressor 3. turn of response when stressor is gone (so you don't hurt yourself or waste energy)

List examples given in class of stress (3):

1. starvation 2. temperature 3. toxic agents (UV, oxidants)-can lead to cell/DNA damage

Myxococcus development proceeds through distinct morphological phases, list them (4):

1. swarming (0hr)- initially starved & looking for food 2. aggregation (7hr)- cells aggregate and pile on top of each other 3. mound formation (12hr)- visible to the eye 4. fruiting body formation (hr)- raised hella, & cells inside turn into myxospores

More than _ genes are part of stationary phase regulon (and sigma factors).

100

Each fruiting body could potentially contain how many bacteria?

10^7 cells in a fruiting body (cells in the middle more likely to become fruiting bodies, nowhere to go. While cells on the outside can take off to look for nutrients)

_% of C. crescentus genes are involved in cell cycle.

20

_ formation is usually in response to an environmental stress or trigger and is when cells attach themselves to a surface by using a polysaccharide capsule/slimy type of material to glue themselves to the surface and remain clumped together.

Biofilm form of differentiation

What controls C. crescentus differentiation?

Cyclic di-GMP levels

What is the control of the heat shock response system (turns the system back off)?

DnaK chaperone/FtsH protease.

_ are a kind of differentiation. When a Bacillus or Clostridium creates an endospore it differentiates & is a dormant cell type that resists more extreme environmental stresses.

Endospores

Reversal frequency controlled by the _ system, analogous to the chemotaxis systems we see in flagellated bacteria.

Frz back and forth movement controlled in a similar way. Just adventurous have reversal frequency??

_ in the cellular starvation pathway are based around determining if I as an individual are starving, and the population starvation pathway is based on coordinating your starvation with everyone else's.

Genes His graduate was on sdeK (histidine kinase responsible for development, he worked on finding the response regulator that went with sdeK).

_ are a cyanobacteria differentiation that are small filaments (dozen cells) that are motile and can glide around (feed & find new area), longer filaments are non-motile (reproduction).

Hormogonia

Is the differentiation of filamentous cyanobacteria terminal or reversible?

Mix of both!

When a biofilm is formed are all of the bacteria the same?

No!

With an increased temperature in the cell the hairpin melts opening the SD sequence for translation. RpoH favors _ binding and expression of heat response genes.

RNAP (More than 250 genes in E. coli - membrane proteins, chaperones)

As ribosomes are stalled we get a protein _ that associated with the ribosome and catalyzes the conversion of GDPorGTP+ATP to (p)ppGpp (second messenger).

RelA

_ makes (p)ppGpp which acts as a master signal in the cell to figure out if it is starving. Myxo starvation & development is induced by aa starvation.

RelA

Cascade of control of the virulence and biofilm formation genes, the Las system controls the _ system.

RhlR

_ is the main convergence point and a way we know a cell is in the stationary phase because it is using this as a sigma factor heavily.

RpoS (σS , σ38)

sRNAs are going to affect the translation of this gene. The cell makes RpoS mRNA (will be moderately stable) and will hang out in the cell & form a hairpin on itself. _ and _ are inaccessible (ribosome can't bind to them). sRNA (DsrA)will be made in response to osmotic stress. A loading protein (chaperone) helps the DsrA bind (annealing) onto the RpoS mRNA. sRNA is targeted for degradation and exonucleases chews away most of the annealed strand which cuts away the hairpin so it opens up the ribosome can bind and start translation.

SD (Shine-Dalgarno) and start codon This is similar to heat shock stress response.

Generation of this signal also depends on protease activity. Name the signal!

Signal B

Cell-surface signal sensed by cell-cell contact (spatial awareness). Orients cells to aggregate properly. Name the signal!

Signal C

Probably influences autolysis. Name the signal!

Signal D

Fatty-acid based signal. Name the signal!

Signal E

What is the main goal for exospores:

We see exospores with actinomycetes, and they are about dispersing the spores. =survival and the germination.

If you knock out the A or S motility will the cell still be able to move?

Yes! Each system has its own genetic control system, so if you take out on system of motility the other still works *spent a long time on this slide

In the 3rd step of dental biofilm formation, fermentative bacteria proliferate & they produce _ that erode dental enamel.

acids

We often see _ cells paving the way and then social motility take over to bring the rest of the group of myxobacteria to where is ended up.

adventurous

In the 1st step of dental biofilm formation, the bacteria are able to attach to tooth surface/pellicle, & resists normal mechanisms of removal (chewing, saliva). Aerobic or anaerobic? Is this 1st step intermittent or constant?

aerobic constant state in our mouths

Other vegetative cells in the filament pass nutrients (ATP, reducing power, sugars) to the heterocyst. The heterocyst then passes back the _ that it is making. =symbiotic relationship between the different cell types of the same species.

ammonium

In the 3rd step of dental biofilm formation, the biofilm thickens, are the deeper areas aerobic or anaerobic?

anaerobic

Some cells will detach over time & can form new _. Some biofilms are semipermanent, some are transient

biofilms

In a stringent response, the cell decreases production of tRNA and rRNA to conserve energy. Protein RelA downregulates protein synthesis through production of pppGpp. Cell increases _ of needed aas.

biosynthesis

_ (secondary messenger) is part of the biofilm control process. Pseudomonas aeruginosa uses it to determine if they want to leave or join the biofilm. in the early formation of the biofilm, this second messenger is made you end up binding and making the adhesin. If it is not made you will be motile and not join the biofilm.

c-di-GMP gene for biofilm production made in 1st quorum system is the enzyme that makes c-di-GMP (Takeaway-quorum sensing can impact a second messenger system)

You can be starved for a specific nutrient or starved in general. You could be requiring aa and vitamins and if you do not get a particular vitamin you be starving for it. General starvation is when you are lacking _.

carbon or energy source (wouldn't be able to do anything with this level of starvation)

aa levels are detected during translation, & when cells are starved for aas the ribosome gets stuck on different codons. Protein synthesis will be unable to proceed because there will not be _ for the ribosome to proceed and it dissociates (works similar to a stop codon).

charged tRNAs

Why do Myxo have rippling behavior when feeding?

could potentially be a mechanism to distribute growth materials that do not normally diffuse well (components of cells being fed on can be large and need to be chopped up and degraded)

A common stress response of bacteria is often to do differentiation and/or development. Differentiation is an adaptive response of cells to their environment, they're going to turn themselves into a _ cell. So due to stress, we have a difference from the vegetative/normal cell leading to this differentiation and/or development. This can take many forms in bacteria.

different-looking/acting (Vegetative cell is "cell", surrounding in image are the responses to stress)

The stringent response can underlie and control a lot of _ responses to stress.

downstream

Myxococcus are motile but they do not have a?

flagella they do make pili-used for 1 type of motility

The gene for the heat shock factor is RpoH (σH , σ32). cells make this rpoH mRNA and leave it sitting around but the protein will not be made all the time due to the _ (heat sensitive) that prevents transcription.

hairpin

What is the function of DnaK?

helps to fold other proteins correctly. If a proteins get unfolded/misfolded from heat stress can refold them into functional forms.

Based on genome sequence there are 7,500 predicted genes, and of those genes 148 are response regulators with roughly the same number of _ because they are often transcribed together in an operon.

histidine kinases

The C. crescentus cells grow and set up a septation ring and the c-di-GMP is produced at the _.

holdfast end of the stalked cell stalked cell=more c-di-GMP swarmer cell=less c-di-GMP

If the Frz system is knocked out in Myxo will they still be able to feed on prey?

if Frz knocked out they cannot do chemotaxis & will be unable to move towards their prey

What does RhlR + AHSL (C4-HSL) increase the expression of (3 gene types)? 2nd quorum system

increase expression of (1) additional virulence genes, (2) RpoS (stationary phase σ), (3) RhlI production-keeps itself going.

What does the LasR + AHSL (3-oxo-C12-HSL) increase the expression of (3 gene types)? 1st quorum system

increase expression of genes for (1) virulence, (2) biofilm production, & (3) RhlR production.

There are more _ in the stationary phase because we have a more error-prone version of the DNA polymerase being made in the stationary phase. maybe you can evolve a new functionality to an enzyme that makes you more efficient and outcompete other cells around you.

mutations more errors=more mutations

Is growth in the motile or nonmotile form?

non-motile, when in biofilm

If we switched to the 2nd quorum system too early you only make a little of the toxin to lyse host cells and it will not be able to do enough damage in order to gather _. By waiting for more cells to be in the biofilm you will have more toxin being made and the desired effect will happen.

nutrients

Second messengers are important in stress responses. They are small molecules produced in a cell in response to a signal (from the first messenger) that is outside the cell (these are often part of signal transduction pathways). We need to amplify signals in ways that are recognizable to the cell and this can be done using second messengers. List 4 examples of second messangers:

often derivatives of nucleotides 1. Cyclic adenosine monophosphate (cAMP). 2. Guanosine tetraphosphate (ppGpp). 3. Guanidine pentaphosphate (pppGpp). 4. Cyclic dimeric GMP (c-di-GMP)/c-di-AMP/cGMP.

A heterocyst is a differentiation formed in cyanobacteria when cells are lacking nitrogen and they synthesize nitrogenase enzyme & degrade photosynthesis machinery. Nitrogen gas is converted to ammonium and this uses a lot of energy and is _ sensitive. It's metabolism is also slowed down.

oxygen

In the 3rd step when the biofilm can extend into pockets between gum and tooth surfaces it leads to?

periodontitis (soft tissue infection)

Motility and heterocyst formation done by cyanobacteria has been co-opted by?

plants! including ferns, bryophytes they create chemicals that attract motile hormogonia to come into contact with plant which will them sequester them into pockets to starve them for nitrogen to convince them to become heterocysts (like 90%). Plants act as filamentous vegetative cells to with the heterocyst.

Development happens when cells are starved for nutrients & requires a large _ working together.

population

Five extracellular signals produced during development, coordinates development and ties together individual cell state to _.

population state

We need signaling to attract and maintain cells in a biofilm, and biofilm formation often requires _.

quorum sensing

RpoS has large cell-wide effects, but the response is tuned by some of the other _ for more specificity of the response.

regulators (like CRP, FhlA) This is a regulatory cascade

What is the term for regulators regulating other regulators?

regulatory cascade

These differentiation events can be _ or _.

reversible (temporary stage) or terminal (this is what you are now)

* Myxo swarms can prey on many microbes in the soil including G+/G-, yeast, fungus, & some phages. Myxococcus secretes various antimicrobial compounds, antibiotics, proteases, digestive enzymes & has a characteristic _ behavior when feeding.

rippling (used the words fun & neat when talking about predation)

We get RpoS to be made and utilized by using lots of _ regulators (can be RNA or DNA based). _ are the usual first step in integrating multiple inputs into a single response

sRNAs response can be modified based on type of stress the cell is dealing with, we can turn on accessory genes.

Each _ responds to a particular type of environmental signal, and redirects cellular physiology and gene expression to respond. The synthesis of these is done by enzymes that we can regulate.

second messenger image shows examples:

What is the term for when signals are converted from one form into another sequentially?

signal transduction

What are akinetes similar to?

spores! akinetes are not as shut off or tough as spores.

The _ phase is a kind of stress response because by it moving into the _ phase metabolism in response to the stress of lower nutrients and higher waste products in the medium.

stationary

One of the first things a cell cares about are aa levels because these are required for protein synthesis (proteins make up most of a cells material). The _ response is what happens when cells detect they are starved for aas, & it stops using aas for things that are unnecessary. Will divert/recycle aas to core needs of the cell in order to make it through a temporary starvation.

stringent

Cells can find ways to manage with temporary _ & _ stresses, have a specific response.

temperature & toxic agent

Endospores are a _ differentiation with a mother and daughter cell where the mother engulfs the daughter and turns it into a spore and mother cell dies. With germination it will turn into a new vegetative cell.

terminal sporulation=terminal changes, no going back

Is the heterocyst differentiation terminal or reversible?

terminal!

If you add c-di-GMP to the swarmer cell during division what happens?

trick them into creating a new offspring that looks like a stalked cell instead, but won't work properly.

_ cyanobacteria cells= normal photosynthetic cyanobacteria cells growing in a filament.

vegetative green cells

Myxobacteria are _-proteobacteria and are generally releated to the other proteobacteria.

δ (delta) They do enough extra stuff to make them interesting, unfortunately for us :_(.

* Heat stress is common so most bacteria have a heat shock response and they have an _ sensor for the response.

σ-factor mRNA (got excited talking about rpoH)