Nutrient uptake (i.e., solute transport) is a cellular process for acquiring molecules from the cell environment that are needed to support cell growth, metabolism and cell maintenance.
- Cells require many types of essential and non-essential nutrients.
- Cells scavenge compounds present in low to high abundance from their environments and accumulate them intracellularly.
- There are two distinct types of nutrient uptake:
- Passive transport. Passive transport does not require cell energy input. It occurs either by the passive diffusion of a molecule across the cell membrane, or by the facilitated diffusion of the molecule aided by a specialized membrane protein.
- Active transport. Active transport of a nutrient requires a dedicated solute transport system and input of cell energy. There are several types of systems that are differentiated by the mechanism of molecule uptake, the energy source, and the types of proteins present. Examples include the ABC-type transporters, symporters, antiporters, and group translocation transporters.
- Solute transport systems are also used to maintain intracellular ion levels, and to export cell waste materials and toxins.
General background on Nutrient Uptake
E. coli, like most microorganisms, thrives in environments that are often limiting for the many types of nutrients needed to support cell growth. To accumulate these molecules the cell employs dedicated nutrient uptake systems called solute transporters. Most of these require energy input in form of the proton motive force or ATP hydrolysis to drive nutrient uptake into the cell.
To accomplish nutrient uptake, the cell must overcome three major obstacles.
First, the cytoplasmic membrane is impermeable to most types of molecules and this restricts their movement across it. Second, the cell must accumulate each nutrient from a generally low concentration present in the surrounding environment, to a much higher level inside the cell. Third, the cell must prevent the entry of harmful molecules that are toxic or potentially harmful while ridding the cell of waste materials. E. coli has acquired through evolution several very elegant solutions to deal with the above obstacles!
The number and types of nutrients used by the cell is large. Some nutrients are essential and the cell cannot survive without them.
For example, certain vitamins, inorganic salts and metals such as phosphate, Mg2+, K+, Cu2+, and Fe2+. The cell also requires other essential compounds that contain carbon, nitrogen and/or sulfur needed to support cell biosynthesis or to generate cellular energy to fuel the above processes.
Many other nutrients are not essential for E. coli cell survival but they are acquired by the cell in order to conserve energy…
that would otherwise be expended for their synthesis. E. coli possesses several hundred distinct nutrient uptake systems. They differ in the type of substrate acquired, the energy required, and their mode of operation. The following examples show how several well studied systems function.
Specific information on E. coli Nutrient Uptake
I. Passive transport across membranes
Entry of solutes into the cell by simple diffusion is generally limited to a few types of molecules since the cytoplasmic membrane forms a hydrophobic barrier to most types of nutrients. The driving force for simple diffusion depends on the solute concentration gradient across the membrane. The molecules will flow from the region of high concentration to the region of lower concentration. Thus, the concentration inside the cell can never be higher than the level outside the membrane.
There are two types of passive transport systems called passive diffusion and facilitated diffusion. They differ in that one type employs a special protein to assist in moving the molecule across the membrane while the other does not.
Facilitated vs Passive Diffusion
The solute molecule shown above (ammonia) enters the cell at a much faster rate via the ammonia-specific facilitator protein compared to passive diffusion.
1. Passive diffusion.
Passive diffusion of a nutrient across the membrane does not require cell energy or any cell proteins. Several types of gasses (e.g. O2, N2, CO2) plus small, uncharged polar molecules (e.g. H2O, glycerol, urea) can pass directly through the cytoplasmic membrane at reasonable rates as they are neither particularly hydrophobic nor hydrophilic in nature. The rate of diffusion of most types of molecules is generally very slow.
The direction of molecule movement across the membrane is determined entirely by the concentration of the molecule on the outside versus the inside of the cell. When the concentration is higher outside, molecules will flow into the cell. Conversely, if the concentration is higher inside, molecules flow out of the cell. In reality, passive diffusion across the cytoplasmic membrane does not generally supply sufficient amounts of required nutrients to support cell growth.
2. Facilitated diffusion.
This process is similar to passive diffusion except that the cell employs a specialized protein embedded in the membrane that “facilitates” the movement of the molecule across it. This allows entry of charged and polar molecules that would otherwise be excluded by the hydrophobic membrane barrier. Without a facilitator protein, highly abundant molecules in the environment could not otherwise cross the membrane.
There are two types of facilitator proteins called carrier proteins and porin proteins. The former are always located in the cytoplasmic membrane and the latter are always located in the outer membrane. Both proteins increase the rate of molecule diffusion across the membranes but they work in different ways.
- Carrier proteins (uniporters)
are embedded in the cytoplasmic membrane and are generally nutrient-specific (i.e., specific for a singe type of molecule). The synthesis of the carrier protein is often regulated by the availability of the molecule transported. Carrier proteins form selective pores that open and close to control passage of that molecule into or out of the cell. Thus, they speed up the rate of molecule movement relative to passive diffusion. Again, this process does not require cellular energy. For example, in E. coli glycerol is transported efficiently across the cytoplasmic membrane by the GlpF permease (GlpF glycerol MIP channel).
Operation of a Uniport System
The potassium ion transporter is an example of a uniport system. This animation demonstrates directional movement of K+ along a gradient and substrate specificity.
- Porin proteins
are embedded in the E. coli outer membrane and facilitate the movement of molecules from the cell exterior to the cell periplasmic space. No cell energy is used and since they operate by simple diffusion. They also allow molecules to flow in the reverse direction. The various porins make up an estimated 50% of the total protein present in the outer membrane.
Porins are divided into two types.
The nonspecific porins allow numerous types of small, hydrophilic and hydrophobic molecules to pass freely across the outer membrane via small pores in the proteins (e.g., the E. coli OmpC and OmpF proteins). However, the size of the molecule must be below ~600 Da in size. Larger molecules cannot traverse the pore because the diameter of the pore is too small for them (~ 8-10 Angstroms).
The substrate-specific porins, as the name suggests, recognize a specific type of molecule. Generally, the molecules are too large to enter by the non-specific porins. Molecules that diffuse through the substrate-specific porins include maltose dextrans, long chain fatty acids, vitamin B12, and the Fe3+-siderophore complexes. Some of these substrate specific porins recognize closely related family of molecules, for example, maltose and the larger maltose dextrans (e.g., MalB).
II. Active transport across the cytoplasmic membrane
The majority of the nutrients required for cell metabolism are taken up by active transport systems. This process is carried out by one or more proteins located in the cytoplasmic membrane or associated with it. All of these systems require the expenditure of cell energy supplied in the form of ATP, the proton motive force, or for some sugar transporters, by the high energy compound, phosphoenylpyruvate (PEP). Active transport systems are highly specific for an individual molecule or class of structurally related compounds.
Since active transport is driven by the expenditure of cell energy, it allows the cell to accumulate molecules to a much higher concentration inside the cell (i.e., the cytoplasm) relative to the outside of the cell (i.e., the periplasm, or cell exterior). The synthesis and/or activity of many of these active transport systems are often regulated depending on the cellular needs.
Types of Active Transport Systems
Active transport systems are divided into three general classes that are named according to their component parts or their mechanism of action. In most cases the solute is transported from the periplasm to the cell interior (i.e., cytoplasm) in an unmodified form. In a few cases the solute is chemically altered during its transfer across the cytoplasmic membrane (e.g., group translocation).
- ABC transporters.
The ATP-Binding Cassette (ABC) transport systems comprise a large class of active transport systems (>200 known in E. coli). They deliver molecules across the cytoplasmic membrane with the concomitant hydrolysis of ATP as the driving force for molecule uptake. Typical substrates include many amino acids, sugars and inorganic ions. Three protein components make up a typical ABC transporter system and include a) a solute binding protein, b) a trans-membrane spanning protein, and c) an ATP hydrolyzing protein that energizes the uptake process. The former protein is located in the cell periplasm, and the second and third form a complex on the cytoplasmic membrane.
In Gram-negative bacteria,
solute binding proteins diffuse freely in the periplasmic space and bind the targeted molecule with very high affinity (i.e., recognizing the molecule at concentrations as low as 10 nM). The binding protein delivers the bound solute to the transmembrane-spanning protein that transports it into the cell by a channel that is selective for only the desired moleucule. It will not allow other types of molecules to enter the cell. The third protein component is an ATP hydrolyzing protein attached to its membrane bound partner at the inner surface in the cytoplasmic membrane. It provides the driving force for solute uptake into the cell. Here, the hydrolysis of ATP to ADP + Pi alters the conformation of the membrane protein component which allows the solute to then enter the cytoplasm. The leucine ABC transport system in E. coli is one well studied example.
The Gram-positive bacteria and the archaea
also possess many ABC-type transport systems. However, they lack an outer membrane like that found in E. coli. To keep their solute binding proteins nearby, these solute binding proteins are tethered to the cytoplasmic membrane via a hydrophobic tail composed of about 25 amino acids. Otherwise, they could be lost to the surrounding medium.
Iron III transport in Gram negative bacteria.
Certain highly specialized ABC transport systems in E. coli function with a cognate substrate specific porin protein. They coordinate acquisition of a specific solute from the cell exterior to bring it into the periplasm, and subsequently into the cytoplasm. One example is the siderophore-mediated iron III (Fe+3) uptake system in E. coli. Iron acquisition is essential for cell growth since this metal is required for the synthesis of many iron-containing proteins including cytochromes and iron-sulfur proteins.
Acquisition of Fe3+ ions is a more complicated process than for uptake of most nutrients
since this form of iron is extremely insoluble and is unable to freely diffuse into the cell in the amounts needed to support cell growth. To overcome this hurdle, many types of bacteria secrete one or more types of iron-binding molecules (i.e., siderophore). For example, a molecule such as hydroxamic acid, citrate, or a more specialized molecule made by E coli called enterobactin. The latter contains multiple catechol rings that tightly binds (i.e., chelates) ferric ions to form a soluble iron protein complex. Hydroxamic acid and citrate also can chelate a molecule of iron.
The Fe3+-enterobactin complex enters the cell periplasm by a substrate-specific outer membrane porin called FhuA. Once the chelate complex is inside, it is then recognized by the cytoplasmic membrane ABC transport system specific for Fe3+-enterobactin (FhuBCD) that in turn transports it to the cytoplasm. After entry, an enzyme named ferric reductase reduces ferric iron (Fe3+) to ferrous iron (Fe2+). This causes the release of the iron molecule from the siderophore so that it can be subsequently incorporated into iron storage and other iron-containing proteins. The newly released enterobactin molecule is exported from the cell and reused.
Operation of ABC-type Transport System
This ABC-type transport system imports solute molecules into the cell. The solute molecule shown in green is bound by the blue periplasmic binding protein which passes the solute to the cognate membrane spanning protein. The energy released by the hydrolysis of ATP to ADP and Pi (inorganic phosphate) drives the solute molecule into the cytoplasm.
The two types of secondary transport systems are defined by the directions of solute and ion movement (i.e., co-transported or antiported).
perform the co-transport of a solute molecule along with the driving force ion. The physiological direction of co-transport is usually into the cell. The lactose permease encoded by the lacY gene is one extensively studied system. It imports one molecule of the sugar lactose (against a proton concentration gradient which is low on the outside to high on the inside). The entry of a single proton provides the energy to uptake one molecule of lactose which is rapidly metabolized after entry into the cell, thus enhancing the rate of solute uptake.
The TRAP transporters ((tri-partite ATP-independent periplasmic transporter) and the TTT transporters (tri-partite tri-carboxylate transporter) constitute a subfamily of the secondary transport system symporters. Both transport systems are composed of three proteins, one of which is a periplasmic substrate binding protein similar to that found in ABC transporters.
The TRAP and TTT transporters appear to have evolved independently as they display no sequence similarity. One example of a TRAP transport system is the E. coli YiaMNO system for 2,3-diketo-L-gulonate uptake. An example of an TTT-type system is the Salmonella typhimurium TctABC transporter for citrate uptake.
transport a solute molecule (either from the outside of the cell to the inside, or in the reverse direction). However, the driving ion molecule (often a proton) moves in the opposite direction (i.e., the opposite of a sympoter). Examples include the exchange of H+ for Na+ ions by the Na+/H+ antiporter, which functions to balance the cytoplasmic pH in E. coli and other bacteria. Some antiporters can import a nutrient while exporting a waste product; e.g. the NarK nitrite transporter then couples the import of the respiratory substrate, nitrate, to the export of the toxic waste product, nitrite.
Operation of a Symport System
The sugar disaccharide (green molecule) transporter is an example of a symport system. Sugar uptake is driven by a proton entering the cell (i.e., by a proton gradient or the pmf).
Operation of an Antiporter System
The Na+/H+ antiport system exchanges Na+ for H+. Sodium export is driven by the proton gradient (pmf).
- The phosphotransferase system (PTS).
In E. coli, the phosphotransferase systems are employed to import several types of sugars (e.g., glucose, fructose, manitol). The high energy phosphate ester bond of PEP is used to drive uptake process where the sugar molecule is covalently modified to a sugar phosphate as the molecule enters the cell.
The E. coli PTS system for glucose is composed of five proteins. They are called HPr, Enzyme I, Enzyme IIA, IIB, and IIC where enzyme IIC is an integral membrane subunit that forms the channel for molecule uptake (i.e., glucose). Enzymes IIA and IIB are attached to Enzyme IIC at the inner surface of the membrane and form a protein complex. Enzyme I and Hpr are both located in the cytoplasm and interact with the Enzyme IIABC complex that together drive glucose into the cell.
The overall transport of glucose seems overly complex but is actually rather simple. Phosphate bond energy derived from PEP is transferred sequentially from protein to protein to protein, and finally onto the glucose molecule to form glucose-6-phosphate.
The order of successive phosphorylation/ dephosphorylation events starts with the HPr protein, and is followed by Enzyme I, Enzyme IIA and then Enzyme IIB. The only protein that is not phosphorylated is Enzyme IIC. Enzyme IIB-phosphate is the direct phosphoryl-donor to glucose.
The conversion of glucose to glucose-6-phosphate is advantageous to the cell! Since the transported molecule is structurally altered, it can no longer leave the cell via Enzyme IIC. Second, the sugar modification generates the first intermediate of the glycolysis pathway. This saves the expenditure of an additional ATP molecule that would otherwise be needed to make G-6-P from glucose using the glycolysis enzyme, glucokinase.
Enzymes IIA, IIB, and IIC are specific for the type of sugar molecule transported (e.g. glucose). Interestingly, E. coli contains at least 10 distinct Enzyme II complexes specific for other sugars (e.g., fructose, mannose, manitol). Expression of the genes for enzymes IIA, IIB and IIC is activated by the presence of its specific substrate. In contrast, the soluble PTS components, Enzyme I and HPr, are not substrate-specific and are used interchangeably by the different sugar specific PTS Enzyme II transporters.
Enzyme IIA also has cell regulatory role! It monitors the cell environment for glucose. If the concentration of glucose decreases, phosphorylated Enzyme IIA subunit accumulates and this activates the membrane-bound adenylate cyclase enzyme. This results in catabolite repression of gene expression due to the buildup of cyclic-AMP in the cell cytoplasm.
- E. coli fatty acid transport.
Transport of long-chain fatty acids into E. coli occurs by a group translocation system distinct from the PTS type system described above. Uptake of the fatty acid molecule presents several challenges since they have very low solubility in aqueous environments and are readily partitioned to the cytoplasmic membrane. In E. coli, the FadL/FadD group translocation system imports long-chain fatty acids using by a three step process. Long-chain fatty acids enter the periplasm via the specialized outer membrane porin protein called FadL. Once inside the periplasm, the fatty acids become protonated which allows them to then diffuse into the cytoplasmic membrane. The proton motive force drives this event.
The FadD protein with a molecule of bound ATP traffics within the cytoplasmic membrane until it recognizes and binds a fatty acid substrate molecule. FadD then covalently attaches a Co-enzyme A molecule to the fatty acid using the energy of ATP hydrolysis. FadD then delivers the fatty acid-CoA molecule into the cytoplasm where it is further metabolized.
The mechanism of solute uptake differs from the ABC-type transporter systems even though both processes use ATP as the energy source. Here, ATP hydrolysis drives the attachment of a Co-enzyme A molecule to the incoming fatty acid rather than phosphorylating it.
Operation of a PTS-type Transport System
The Glucose-specific PTS transporter is an example of a group translocation system. Here, glucose (the green hexagon) located in the cell periplasm space is bound by the membrane protein Enzyme IIC. Energy from PEP is used to drive glucose uptake via a phosphorylation cascade where glucose is ultimately phosphorylated as it enters the cell.
- Nutrients enter the cell via passive or active transport mechanisms.
- Passive transport mechanisms do not require energy input and involve simple diffusion of solutes across the membrane.
- Passive transport can occur with or without involvement of a protein to facilitate diffusion.
- Active transporters always require energy input and involve dedicated transport proteins embedded in the cytoplasmic membrane.
- The three general types of active transporters include the ABC transporters, the secondary transporters and the group translocation systems.
- ABC transporters employ a periplasmic solute binding protein, a membrane intrinsic transport protein and an ATP hydrolyzing protein to drive solute uptake.
- Secondary transporters import solutes either by co-transport (symport) or by molecule exchange (antiport of an ion). The driving force for most secondary transporters is the proton motive force; alternatively, the gradient of the co- or anti- transported ion drives transport.
- Group translocation systems modify the solute upon cell entry. PEP or ATP provide the energy for the modification reaction.
- Molecules are accumulated against an increasing concentration gradient during active transport.
Authored by Robert Gunsalus and Imke Schroeder
©The Escherichia coli Student Portal
This project acknowledges support from:
Peter Karp and coworkers at EcoCyc.org
NIH Grant Award GM077678 to SRI, International
Animations from surfrender
The UCLA Department of MIMG