4 - Bacterial Culture, Growth and Development Flashcards
What drives development and cellular differentiation?
Starvation (e.g, the formation of spores)
When will the bacteria’s growth be exponential?
If we place a typical bacterium like E. coli on a resource-rich medium with optimal conditions
Why does logarithmic growth not occur in the real world?
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
What does food provide?
- Carbon source
- Energy
- Electron
- Macro and micro nutrients
Auto- and Hetero-
- Auto-: CO2 is fixed to make all organic molecules.
* Hetero-: organic molecules are imported and metabolized
Photo- and Chemo-
- Photo-: Light absorption captures energy.
* Chemo-: Chemical electron donors are oxidized.
Litho- and Organo-
- Litho-: inorganic molecules donate electrons.
* Organo-: organic molecules donate electrons
What is the difference between micro and macro nutrients?
The amount we need of them
• Macro-nutrients: carbon, nitrogen, phosphorus, sulfur, calcium.
• Micro-nutrients: iron, magnesium, zinc, etc
What are the 2 general types of media that bacteria can grow on in labs?
Luria Bertani media (LB) and M9 media
What is LB media also called?
Complex/complete media. It is complex in the sense that raw products have come from other organisms
What kind of bacteria can LB media grow?
Both gram positive and gram negative bacteria
How do we form an LB media?
(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
What is M9 media also called?
Defines/minimal media
What do we do in M9 media?
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.
What is contained in M9 media?
Glucose sodium potassium nitrogen NaCl iron water
What kind of bacteria can grow in M9 media?
Only gram negative
What is batch culture growth?
Batch culture growth refers to a technique used to grow microorganisms where only limited supply of nutrients for growth is provided
What are the three phases in batch culture growth?
Lag
Log
Stationary phase
What kind of culture do most bacteria live in in natural cirumstances?
Continuous culture
What kind of environment do bacteria in our gut live in
Continuous culture
What are the two types of continuous culture states?
(1) Turbi-stat
(2) Chemo-stat
What do we keep constant in Turbi-stat
Turbidity or cell density
Why does turbi-stat result in more exponential growth?
Because the bacteria are constantly being provided with a fresh supply of nutrients
How does turbi-stat work?
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).
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)
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.
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.
What are the main properties of chemo-stat?
Growth is determined by flow rate
Cell density is determined by media
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
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).
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
What is facilitated diffusion?
The movement of molecules down their concentration gradient
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.
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.
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.
What do antiporters and symporters do?
Transport nutrients by a coupled transport with driving ions (H+ or Na+) that move down their concentration gradient.
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
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
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.
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.
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.
