4 - Bacterial Culture, Growth and Development Flashcards
1
Q
What drives development and cellular differentiation?
A
Starvation (e.g, the formation of spores)
2
Q
When will the bacteria’s growth be exponential?
A
If we place a typical bacterium like E. coli on a resource-rich medium with optimal conditions
3
Q
Why does logarithmic growth not occur in the real world?
A
This logarithmic growth does not occur on a long period of time, in fact it is kind of rare because, most of the time, bacteria are starving
4
Q
What does food provide?
A
- Carbon source
- Energy
- Electron
- Macro and micro nutrients
5
Q
Auto- and Hetero-
A
- Auto-: CO2 is fixed to make all organic molecules.
* Hetero-: organic molecules are imported and metabolized
6
Q
Photo- and Chemo-
A
- Photo-: Light absorption captures energy.
* Chemo-: Chemical electron donors are oxidized.
7
Q
Litho- and Organo-
A
- Litho-: inorganic molecules donate electrons.
* Organo-: organic molecules donate electrons
8
Q
What is the difference between micro and macro nutrients?
A
The amount we need of them
• Macro-nutrients: carbon, nitrogen, phosphorus, sulfur, calcium.
• Micro-nutrients: iron, magnesium, zinc, etc
9
Q
What are the 2 general types of media that bacteria can grow on in labs?
A
Luria Bertani media (LB) and M9 media
10
Q
What is LB media also called?
A
Complex/complete media. It is complex in the sense that raw products have come from other organisms
11
Q
What kind of bacteria can LB media grow?
A
Both gram positive and gram negative bacteria
12
Q
How do we form an LB media?
A
(1) 10g of Bacto trypton (derived from milk)
(2) 5g of Bacto yeast extract
Yeast extract generated by putting yeast culture into blender in order to release all the nutrients. This extract will be used to create a complete media that will provide the bacteria with more than enough supply of nutrients to survive.
(3) 10g of NaCl
(4) 1L of water
(5) pH is 7
(6) Heat-sterilize the whole thing
13
Q
What is M9 media also called?
A
Defines/minimal media
14
Q
What do we do in M9 media?
A
Here we are adding individual chemicals (macro- and micro- nutrients).
The media can lack a certain nutrient. This can enable the selection of auxotrophs, which are microorganisms that are deficient in a gene that is involved in a certain biochemical pathway. For example, if we want to determine whether bacterial strains on a master plate are deficient in gene A, which is involved in metabolizing nutrient X. We could create an M9 media that lacks nutrient X and then plate the different strains onto this media and determine which ones do not grow: these will be the ones that are deficient in gene A and could, therefore, not metabolize nutrient X.
15
Q
What is contained in M9 media?
A
Glucose sodium potassium nitrogen NaCl iron water
16
Q
What kind of bacteria can grow in M9 media?
A
Only gram negative
17
Q
What is batch culture growth?
A
Batch culture growth refers to a technique used to grow microorganisms where only limited supply of nutrients for growth is provided
18
Q
What are the three phases in batch culture growth?
A
Lag
Log
Stationary phase
19
Q
What kind of culture do most bacteria live in in natural cirumstances?
A
Continuous culture
20
Q
What kind of environment do bacteria in our gut live in
A
Continuous culture
21
Q
What are the two types of continuous culture states?
A
(1) Turbi-stat
(2) Chemo-stat
22
Q
What do we keep constant in Turbi-stat
A
Turbidity or cell density
23
Q
Why does turbi-stat result in more exponential growth?
A
Because the bacteria are constantly being provided with a fresh supply of nutrients
24
Q
How does turbi-stat work?
A
We dilute the bacteria with fresh replete medium as they grow. We dilute it at a certain rate to maintain constant turbidity (constant cell density). Replete media is a media that gives the bacteria everything that they need to grow. So, if we stop supplying media to the growing cells, we find ourselves in a batch culture. Here, bacteria don’t have to compete for nutrients because they are constantly diluted (this is not what happens in nature).
25
What are the properties of turbi-stat?
Growth rate determined by media
| Cell density determined by flow rate (faster you dilute the lower the cell density)
26
What is the main principle in Chemo-stat
The nutrients that we provide our bacteria with are actually limiting. When we stop supplying the media in the case of chemo-state, bacteria will stop growth instantly. To keep the bacteria alive, we need to constantly add fresh media which contains fresh limiting glucose. In the case of chemo-state, we can adjust the growth rate of bacteria. We do that by adjusting the flow rate of fresh limiting nutrients. Chemo-state stimulates more closely what happens in continuous culture where nutrients are limiting but are constantly flowing in.
27
How can be study how bacteria respond to limiting nutrients?
We have to do it in chemo-state since the chemo-state reflects true environmental conditions that bacteria experience, as there is not always an endless supply of nutrients available to them at all time.
28
What are the main properties of chemo-stat?
Growth is determined by flow rate
| Cell density is determined by media
29
Imagine the following experiment: a chemo-stat is used with a maltose medium. Cell density stays constant over time and then suddenly jumps up where it again stays constant. Why did cell density rise?
Because cell utilization of maltose increased
30
Why is 1/3 of the bacterial genome devoted to membrane proteins?
Because import of nutrients into the cytoplasm is vital. All bacteria must uptake nutrients. Nutrient uptake is vital when nutrients are limiting (in chemo-stat or most places).
31
What are the 5 transport systems that enable nutrient transport across membranes?
(1) Facilitated diffusion
(2) Antiporters and symporters
(3) ABC transporters
(4) PTS
(5) Ton-dependent systems
32
What is facilitated diffusion?
The movement of molecules down their concentration gradient
33
What is GlpF?
A transmembrane protein that mediates facilitated diffusion. Specifically, GlpF facilitates glycerol transport from outside environments to the cytoplasm. The transmembrane part of GlpF is hydrophobic alpha-helices and found in the plasma membrane.
34
How does GlpF work?
It is the concentration gradient that will drive glycerol inside the cell. No energy is needed. GlpF is a gated channel. First, glycerol will enter from the extracellular space. Once glycerol is inside GlpF, a conformational change will occur. The extracellular face will close and the cytoplasmic face will open, leading to the release of glycerol inside the cell.
35
GlpF vs. OmpF
GlpF (inner membrane protein) is more specific than OmpF (outer membrane protein F/porins) because OmpF are basically just holes. OmpF therefore enable the uptake of multiple nutrients, which could be useful in the case of starvation for example, but the fact that OmpF are non-specific allows antibiotics to enter, which is harmful to the cell. OmpF should not be in the inner membrane because they are just holes and you want the inner membrane to be sealed.
36
What do antiporters and symporters do?
Transport nutrients by a coupled transport with driving ions (H+ or Na+) that move down their concentration gradient.
37
How do symporters work?
A symporter is an integral membrane protein that is involved in the transport of many different types of molecules. Symporter transports 2 molecules at the same time and in the same direction (both going in or both going out of the cell). One molecule is crossing the membrane down its concentration or electric gradient (no E required: facilitated diffusion). The energy released from the transport of the first molecule drives the transport of the second molecule that is going against its concentration gradient
38
What are some examples of symporters?
(1) LacY symporter. LacY imports lactose using proton motor force: H+ flows into cell down their concentration gradient, providing the E required to import lactose. Thus, the symporter is electrogenic (increase in + charge inside the cell). There is then a H+/Na+ antiporter that uses the E from Na+ flowing into the cell down its concentration gradient to pump H+ back out. Since the overall charge across the membrane does not change, this antiporter is electroneutral.
(2) Sodium and Leucine
39
How do antiporters work?
An antiporter is an integral membrane protein that is involved in the transport of many different types of molecules. One species of solute moves along its electrochemical gradient, allowing a different species to move against its electrochemical gradient.
40
How are antiporters different from symporters?
In the case of the antiporters, both molecules are not transported in the same direction, i.e. one enters the cell and the other one exits the cell.
41
What are ABC transporters?
ATP binding casettes. These transporters use the energy from ATP hydrolysis to move solutes against their concentration gradient and they are found in the inner membrane. ABC transporters are ubiquitous for species of the 3 kingdoms of life.
42
How do ABC transporters work?
The solute binds to its specific solute-binding protein in the periplasmic space. Maltose as the solute has been the best studied system. This complex will then bind to the periplasmic side of the ABC transporter. The binding triggers a conformational change that activates the ATPase activity of one of the components of the transporter, causing it to hydrolyze ATP. ATP hydrolysis then leads to the opening of channel and the movement of the solute into the cell.
43
Can ABC transporters work backwards?
Yes
44
What is the phosphotransferase system (PTS) and how does it work?
These are also called group translocated systems and are found in the inner membrane (PM). PTS phosphorylate sugars during transport. Glucose is phosphorylated as it gets actively transported into the cell. When the sugar gets phosphorylated, it will get stuck in the cytoplasm. This system is not used in humans - it is bacteria specific.
45
What are the advantages of the PTS?
(1) It is very efficient.
(2) Once we get the sugar in, it is ready for metabolism because it is phosphorylated. Normally it will get phosphorylated by hexokinase using ATP but here the sugar is already phosphorylated.
46
What kind of nutrients does PTS, antiporters, symporters, ABC, and facilitated diffusion import?
Macro-nutrients
47
What is the problem associated with importing micro-nutrients?
Their concentration in the environment is very low. This suggests that the systems used to import these micro-nutrients should have very high affinities.
48
What does the import of micro-nutrients involve?
Ton-dependent receptors
49
What are ton-dependent systems (in OM, gram-negative bacteria)?
High-affinity, stereo-selective outer membrane proteins.
Ton = general IMPs plugged into highly selecive OMPs.
Ton proteins transduce the proton motive force from IM to OM.
50
What are siderophores?
E. coli will synthesize and secrete a siderophore called enterochelin. Siderophores are small, high-affinity iron-chelating compounds secreted by bacteria. Siderophores are amongst the strongest soluble Fe3+ binding agents known.
51
How to ton-dependent systems work?
(1) Enterochelin will bind ferric iron (Fe3+) forming an Enterochelin-iron complex.
(2) This complex will get transported into the periplasmic space through a protein called FepA.
(3) The energy used to drive the transport of the Enterochelin-iron complex originates from the proton motive force, which is the movement of ions across the membrane, down their concertation gradient.
(4) In the periplasmic space, the Enterochelin-iron complex will get bound to a protein called FepB.
(5) FepB will drive the Enterochelin-iron complex to an ABC transporter. The ABC transporter is made from 3 integral membrane proteins: FepC, D, and G.
(6) The ABC transporter will use the energy from the hydrolysis of ATP to transport the Enterochelin-iron complex into the cytoplasm.
(7) In the cytoplasm, ferric iron Fe3+ is released from the enterochelin and it is reduced to Fe2+, which is the form of iron used by the cell.
52
What is FepA?
An integral OM protein involved in the active transport of Enterochelin-iron complex.
53
What is the difference between FepA and OMP porins?
FepA uses active transport whilst porins use passive transport
54
Are ton-dependent transporters exclusive to iron transport?
No, there are vast families of ton-dependent outer membrane proteins in bacteria for the transport of B12, amino acids, vitamins, etc.
55
How many iron uptake systems does E.coli have?
4
56
What is the serial dilution method calculation?
dilution = volume of sample/(volume of sample + volume of diluent)
57
Historical growth experiment
Measure how many cells of Staph. aureus are alive in a flask. Penicillin was added and again the number of viable cells were measured. The numbers had dropped however there were some cells that survived. This was because cells were in different states. Some grew fast whilst others grew slow. Some had a slower metabolism which made them resistant to the pencillin. These cells switch from slow growth to very fast growth
58
What is the difference between inner membrane and outer membrane transport systems?
Inner membrane proteins have alpha helices whilst outer membrane proteins (which are only in gram negative bacteria) have beta pleated sheets
59
Examples of IMPs?
- GlpF
- Symporter
- Antiporter
- ABC transporter
- PTS
- Aquaporins
60
Examples of OMPs?
- Porins
| - Ton-dependent system
61
What is a biofilm?
Any group of microorganisms in which cells stick to each other and a surface. They are where most microorganisms live as they feed and get protection here. These adherent cells become embedded within a slimy extracellular matrix composed of extracellular polymeric substances (EPS).
62
What are extracellular polymeric substances?
Natural polymers of high molecular weight secreted by microorganisms into their environment. EPSs establish the functional and structural integrity of biofilms, and are considered the fundamental component that determines the physiochemical properties of a biofilm.
63
How do bacteria decide to switch from swimming to sticking and forming biofilms?
Inside the bacterial cells, there is a signaling molecule that helps dictate this decision. This molecule is called cdiGMP: a global biofilm signal molecule. There are enzymes in bacterial cells that will link 2 GTP molecules head to tail and form a cyclic diGMP.
64
How does the level of cdiGMP decide whether cells swim or stick?
Low levels of cdiGMP = cells are swimming
| High levels of cdiGMP = cells settle down and form biofilms
65
What are biofilms an example of?
Bacterial differentiation. Bacterial cells forming biofilms have a different set of genes expressed compared to bacteria that are swimming
66
Caulobacter crescentus
When these bacteria start forming biofilms, they eject their flagella. Some signals (cdiGMP) will activate a kinase. This kinase will activate a protease. This protease will cleave a key component in the ring which helps eject the flagella
67
What are other molecules involved in biofilm signaling?
ppGpp = starvation alarm
| Quorum sensing = cell density triggers
68
What are Cyanobacteria?
Bacteria that usually grow on the surface of water. They are photosynthetic prokaryotes (photo-autotrophs) and grow as filaments.
69
Why do cyanobacteria differentiate?
Most cells in the filaments are photosynthetic cells (light green in figure). When nitrogen becomes scarce, cyanobacteria can’t grow. If they can’t get nitrogen from the water, they will get it from the air through a process called nitrogen fixation. During starvation, some of these cells will differentiate into non-photosynthetic cells called heterocysts (dark green in figure). So differentiation is prompted by nitrogen starvation.
70
What are nitrogenase?
In the heterocysts, an oxygen-sensitive enzyme called Nitrogenase will carry out the nitrogen fixation reaction. Nitrogenase will fix nitrogen and make ammonia. Nitrogenase needs to be present in a cell that is impermeable to oxygen
71
How is distance conserved between heterocysts?
In every 10 photosynthetic cells, we find a heterocyst when nitrogen is scarce. One heterocyst cell will send out specific signals to close cells inhibiting them from differentiating to heterocysts
72
How is the Cyanobacteria differentiation response advantageous
It results in a symbiotic relationship where some cells making carbon and others making nitrogen and they share these nutrients. So we have a nitrogen carbon sharing system
73
What are Myxococcus xanthus?
A gram negative bacterium which secrete digestive enzymes (proteases). They use pili to push and pull themselves as well as to communicate.
74
Why do Myxococcus xanthus differentiate from motile cells into dormant spores?
When they start starving, several Myxococci (appx 100,000 cells aggregate) will group together and form a fruiting body and, in that fruiting body, the sporulation program is induced. So amino acid starvation is what prompts differentiation. While being clustered, Myxococci will start making spores and secreting specific substances to keep those spores attached together.
75
Why do spores aggregate as fruiting bodies?
To get more stability and efficiency. o Myxococci need to group together so that their secreted digestive enzyme is in high concentration and can actually kill bacteria and fungi for food (competitive bacteria)
76
What is the differentiation response in Myxococcus xanthus?
o Fruiting body formation
o Motile rods change into glued round spored
o They use type IV pili
o Cell specialization is 1/3 spores and 2/3 support
77
How is the differentiation response in Myxococcus xanthus advantageous?
The fruiting body structure is important because these bacteium eat together and, so, must travel together and the fruiting body ensures this.
78
What are streptomyces?
A soil gram-positive bacterium.
79
Why do streptomyces differentiate from non-motile bacteria to spores?
When conditions are favorable, spores will germinate and form roots. These roots will grow in the soil and become larger and larger. Nucleoids will divide but the cytoplasm is continuous in this roots network. These roots will absorb nutrients in the soil. When nutrients run out from the soil, the cells will differentiate. So here again, what prompts differentiation is starvation
80
What happens during differentiation in streptomyces?
Instead of growing in the soil, they will grow upwards to the air forming areal hyphae. Later, in the areal hyphae, cells will start forming septa (septa=location where cell division occurs) to compartmentalize and separate their nucleoid into individual cells, forming spores.Those spores will get released and they will colonize new soil.
81
What are bld genes?
Mutations in these genes will prevent the soil bacteria from forming areal hyphae. These mutated bacteria will only form a root system.
82
What are whi genes?
When spores start to form, they have a white color and as their development goes further, they will start to have a dark color. Mutations in whi genes will prevent further development of the spores, keeping them white.
83
Why is the differentiation response in streptomyces advantageous?
Streptomyces produce a large number of antibiotics. They secrete a lot of antibiotics to protects themselves from other bacteria and to kill other bacteria, thereby reducing competition. Areal hyphae also contain droplets of antibiotics.
84
How do steptomyces grow?
Streptomyces only have lateral cell wall growth decoupled from separation
85
What is the sporulation gene/protein in streptomyces?
FtsZ
86
What are the two types of spores?
Exo-spores - 1 cell makes 1 spore
| Endo-spores - 2 cells make 1 spore
87
What is the idea behing endo-spores?
While one cell is transforming itself into a spore, the other cell is building the spore from the outside. Endospores are the most resilient forms of life known
88
What is Bacillus subtilis?
A gram-positive bacteria that forms endospores. These are vegetative cells (cells that are actively growing rather than forming spores).They have a symmetrical division and form filaments. When they starve, they will induce endospore formation (unlike E.coli)
89
How are endo-spores formed in Bacillus subtilis?
(1) Once they decide to form endospores (complicated decision process), they will replicate their DNA and then divide asymmetrically (the septum will form near one pole).
(2) This means that one compartment will be smaller than the other.
(3) The smaller compartment will become the endospore.
(4) The larger compartment (referred to as the mother cell) will then engulf the smaller compartment (referred to as the forespore).
(5) After engulfment, the chromosome of the mother cell will disintegrate.
(6) The forespore will then develop a cortex, which is a layer of peptidoglycan between the original forespore membrane and the membrane from the mother cell.
(7) Coat proteins will then be deposited on the outer membrane (mother membrane).
(8) Dipicolinic acid and calcium are then incorporated into the spore coat.
(9) The mother cell will then release the spore.
90
What is Stage I of sporulation?
Septum forms near one pole. DNA replicates and extends into an axial filament.
91
What is Stage II of sporulation?
Septum separates forespore from mother cell. DNA pumped through septum until each compartment gets a chromsome.
92
What is Stage III of sporulation?
Mother cell engulfs the forespore, surrounding it with a second membrane
93
What is Stage IV of sporulation?
Chromsome of mother cell disintegrates
94
What is Stave V of sporulation?
Forespore develops a cortex layer of peptidoglycan between original forespore membrane and the membrane from the mother cell. Coat proteins deposited on outer membrane.
95
What is Stage VI of sporulation?
Dipicolinic acid is synthesized, and calcium is incorporated into the spore coat.
96
What is Stage VII of sporulation?
Mother cell releases spore
97
What are sigma factors?
There are several signals being transmitted between the mother and the forespore. Those signals involve several specific sigma factors. Sigma factors are enzymes that will bind to RNA polymerase and directing it to different promoters
98
Do all Bacillus subtilis sporulate?
No. The degraded genes from the mother cell can be incorporated by other Bacillus subtilis that don’t sporulate, after the mother cell bursts open and releases the spores.The released mother cell DNA permits genetic exchange
99
What is programmed variation?
Which cells will sporulate and which will not
100
What modes of differentiation do Bacillus subtilis exhibit?
Asymmetric cell division, sporulation and programmed variation
101
What are the advantages of endo-sporulation?
Domancy and super-durability
102
What does sporulation involve?
A committee of Pi related proteins. Sporulating is not just a switch but a process.
103
When do Bacillus subtilis decide to sporulate using quorum sensing?
When food is lowe and cell density is high.
104
What are two example of bacillus subtilis being a model for other bacteria?
(1) Epulopiscium fishelsoni - Have a strange division where they split on the side. Also have asymmetric cell division but you divide at both poles (engulf at both poles)
(2) Gut bacteria hair balls - Found in guts of mice and newborn infants. Grows reminiscent of what we learn with bacillus subtilis. Cells grow in chains and divide symmetrically. Eventually switch to asymmetric cell division and smallest cells engulfed by larger cells. Inside they grow and differentiate into hook forms and hook forms released and can float around or reattach
