Active transport- Group Translocation, Iron Uptake

 Group Translocation

In active transport, solute molecules move across a membrane without modification. Many procaryotes also take up molecules by group translocation, a process in which a molecule is transported into the cell while being chemically altered. The best-known group translocation system is the phosphoenolpyruvate: sugar phosphotransferase system (PTS). It transports a variety of sugars into procaryotic cells while phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor.

 PEP + sugar (outside) → pyruvate + sugar -P (inside)

 The PTS in E. coli and Salmonella typhimurium,  consists of two enzymes and a low molecular weight heat-stable protein (HPr).

l  HPr and enzyme I (EI) are cytoplasmic.

l  Enzyme II (EII) is  composed of three subunits or domains- EIIA  is cytoplasmic and soluble. EIIB also is hydrophilic but frequently is attached to EIIC, a hydrophobic protein that is embedded in the membrane.

 

Group Translocation: Bacterial PTS Transport

1. A high-energy phosphate is transferred from PEP to enzyme II with the aid of enzyme I and HPr .

2. A sugar molecule is phosphorylated as it is carried across the membrane by enzyme II.

 Enzyme II transports only specific sugars and varies with PTS, whereas enzyme I and HPr are common to all PTSs. PTSs are widely distributed in procaryotes.

 Except for some species of Bacillus that have both glycolysis and the phosphotransferase system, aerobic bacteria seem to lack PTSs.

Members of the genera Escherichia, Salmonella, Staphylococcus, and other facultatively anaerobic bacteria have phosphotransferase systems;

Some obligately anaerobic bacteria (e.g., Clostridium) also have PTSs. 

Many carbohydrates are transported by these systems. E. coli takes up glucose, fructose, mannitol, sucrose, N- acetylglucosamine, cellobiose, and other carbohydrates by group translocation.

 Iron Uptake 

Almost all microorganisms require iron for use in cytochromes and many enzymes. Iron uptake is made difficult by the extreme insolubility of ferric iron (Fe³⁺) Little free iron is available for transport. Many bacteria and fungi have overcome this difficulty by secreting siderophores [Greek for iron bearers]. 

Siderophores are low molecular weight molecules that are able to complex with ferric iron and supply it to the cell. These iron-transport molecules are normally either hydroxamates, phenolates or catecholates. Ferrichrome is a hydroxamate produced by many fungi; Enterobactin is the catecholate formed by E. coli.

Microorganisms secrete siderophores when little iron is available in the medium. Once the iron-siderophore complex has reached the cell surface, it binds to a siderophore-receptor protein. Then the iron is either released to enter the cell directly or the whole iron-siderophore complex is transported inside by an ABC transporter. After the iron has entered the cell, it is reduced to the ferrous form (Fe²⁺).

Iron is so crucial to microorganisms that they may use more than one route of iron uptake to ensure an adequate supply.

l  Electrogenic transport- Done by transport proteins/carriers that generate voltage across a membrane by the transfer of a charge, e.g., Na+/K+ATPase exchanges 3Na + for 2K + 

        A proton pump (H+) is the major electrogenic pump in plants, bacteria, and fungi. - Voltages created by electrogenic pumps are sources of potential energy available to do cellular work.

l  Electroneutral transport- where carriers exchange an equal number of charged particles or transport uncharged molecules

l  Porins – channels to transport larger molecules eg., aquaporin- allows water to cross the plasma membrane

l  Permeases – function more like an enzyme. Binds the substrate and then undergoes a conformation change which causes the carrier to release the substrate to the other side. Ex. lactose permeases 

Active Transport-Proton gradient driven Membrane Transport

 2. Proton gradient driven Membrane Transport

            Bacteria also use proton gradients generated during electron transport to drive active transport. The membrane transport proteins responsible for this process lack special periplasmic solute binding proteins.

The lactose permease of E. coli is a well-studied example. The permease is a single protein having a molecular weight of about 30,000. It transports a lactose molecule inward as a proton simultaneously enters the cell (a higher concentration of protons is maintained outside the membrane by electron transport chain activity). Such linked transport of two substances in the same direction is called symport.

                  

Here, energy stored as a proton gradient drives solute transport. The binding of a proton to the transport protein increases its affinity for the solute and transports it.

            E. coli also uses proton symport to take up amino acids and organic acids like succinate and malate.

 

Active Transport Using Proton and Sodium Gradients-

A proton gradient also can power active transport indirectly, often through the formation of a sodium ion gradient. 

For example, an E. coli sodium transport system pumps sodium outward in response to the inward movement of protons. Such linked transport in which the transported substances move in opposite directions is termed antiport. 

The sodium gradient generated by this proton antiport system then drives the uptake of sugars and amino acids. A sodium ion could attach to a carrier protein, causing it to change shape. The carrier would then bind the sugar or amino acid tightly and orient its binding sites toward the cell interior. Because of the low intracellular sodium concentration, the sodium ion would dissociate from the carrier, and the other molecule would follow. 

E. coli transport proteins carry the sugar melibiose and the amino acid glutamate when sodium simultaneously moves inward, this is Sodium symport or cotransport..

1 . Protons (H+) are pumped to the outside of the plasma membrane during electron transport.

2. The proton gradient drives sodium ion (Na+) expulsion by an antiport mechanism.

3. The shape of the solute binding site changes, and it binds the solute (e.g., a sugar or amino acid). Sodium binds to the carrier protein complex.

4. The carrier’s conformation then alters so that sodium is released on the inside of the membrane. This is followed by solute dissociation from the carrier (a symport mechanism).

     Often a microorganism has more than one transport system for each nutrient. E. coli has at least five transport systems for the sugar galactose, three systems each for the amino acids glutamate and leucine, and two potassium transport complexes. 

When there are several transport systems for the same substance, the systems differ in such properties as their energy source, their affinity for the solute transported, and the nature of their regulation. This diversity gives the organism an added competitive advantage in a variable environment.

Factors influencing growth and distributions-temperature, pH, pressure, salinity

                              

                                 

Transport of nutrients by bacteria - Active Transport

Diffusion and facilitated diffusion can efficiently move molecules to the interior when the solute concentration is higher on the outside of the cell, they cannot take up solutes that are already more concentrated within the cell (i.e., against a concentration gradient). Microorganisms often live in habitats characterized by very dilute nutrient sources, and, they must be able to transport and concentrate these nutrients against a concentration gradient.

Microbes use two important transport processes in such situations: active transport and group translocation. Both are energy-dependent processes.

Active transport is the transport of solute molecules to higher concentrations, or against a concentration gradient, with the use of metabolic energy input. Because active transport involves protein carrier activity, it resembles facilitated diffusion in some ways. The carrier proteins or permeases bind particular solutes with great specificity for the molecules transported.

Similar solute molecules can compete for the same carrier protein in both facilitated diffusion and active transport. Active transport is also characterized by the carrier saturation effect at high solute concentrations

However, active transport differs from facilitated diffusion in its use of metabolic energy and in its ability to concentrate substances. Metabolic inhibitors that block energy production will inhibit active transport but will not affect facilitated diffusion (at least for a short time).

 

1. ATP driven Binding protein transport

Binding protein transport systems or ATP-binding cassette transporters (ABC transporters) are active in bacteria, archaea, and eukaryotes. Usually these transporters consist of two hydrophobic membrane-spanning domains. These are associated with two nucleotide-binding domains on their cytoplasmic surfaces . 

The membrane-spanning domains form a pore in the membrane and the nucleotide-binding domains bind and hydrolyze ATP to drive uptake.

ABC transporters use substrate binding proteins, which are located in the periplasmic space of gram-negative bacteria or are attached to membrane lipids on gram-positive plasma membrane. These binding proteins, bind the molecule to be transported and then interact with the membrane transport proteins to move the solute molecule inside the cell. 

                ABC Transporter Function.

(1) The solute binding protein binds the substrate to be transported and approaches the ABC transporter complex.

(2) The solute binding protein attaches to the transporter and releases the substrate, which is moved across the membrane with the aid of ATP hydrolysis.

E. coli transports a variety of sugars (arabinose, maltose, galactose, ribose) and amino acids (glutamate, histidine, leucine) by this mechanism.

Substances entering gram-negative bacteria must pass through the outer membrane before ABC transporters and other active transport systems can take action. There are several ways in which this is done. 

• When the substance is small, a generalized porin protein such as OmpF can be used
• larger molecules require specialized porins. 
• In some cases (e.g., for uptake of iron and vitamin B12), specialized high-affinity outer membrane receptors and transporters are used.

 

 Eucaryotic ABC transporters are sometimes of great medical importance. Some tumor cells pump drugs out using these transporters. Cystic fibrosis results from a mutation that inactivates an ABC transporter that acts as a chloride ion channel in the lungs.

Neisseria gonorrhoeae- Antigenic/Virulence Factors, Resistance, Epidemiology

 Antigenic/Virulence Factors

 

•  Pili - Are hair like structures extending from the surface - made up of pilin proteins -                     -Pilin proteins are antigenically different in almost all strains- A single strain can produce several antigenically distinct pili. Piliated gonococci are usually virulent, whereas non-piliated strains are avirulent 

• Lipooligosaccharide – Outer membrane contains LOS-lipooligosaccharide (endotoxin) – responsible for toxicity 

• Outer membrane Proteins – many different proteins 

• Protein I (por) – Forms pore on surface. Each strain expresses one type of protein I. It helps in serotyping of gonococci. Two variants of protein I – IA & IB. Any one strain carries either IA or IB but not both. 24 serovars of type IA & 32 serovars of type IB. 
• Protein II (opa) – Opacity associated outer membrane protein (OPA). Help in attachment to host cell. Strains with OPA protein form opaque colonies.  
• Protein III – is associated with protein I in the formation of pores on the cell surface &  plays a role in the exchange of molecules across the outer membrane.

• IgA1 protease - The main host defenses against gonococci are antibodies (IgA and IgG), complement, and neutrophils. • IgA protease degrades and inactivates IgA which plays a major role in mucosal defense. 

•  Transferrin (Iron binding proteins) specifically bind and internalize iron from host‐derived proteins, including transferrin, lactoferrin

•  Plasmids-Gonococci contains several cryptic plasmids –  transmissible plasmids contain genes that code for beta lactamase which causes resistance to penicillin. 

Resistance

• Very delicate organism - Readily killed by drying, heat & antiseptic 
• Strict parasite & dies in 1 – 2 hours in exudate outside the body. 
• In culture, the coccus dies in 3 – 4 days but survives in slant culture at 35⁰C if kept under sterile paraffin oil. 
• Cultures – preserved for years if frozen quickly & stored at – ^- 70⁰C.

Epidemiology

• ·   Exclusively a human disease- no natural infection in animals. Experimental infection in chimpanzees (urethral inoculation)  and mice (intracerebral inoculation)
• ·         Humans-only source of infection
• ·         Asymptomatic carriage in women- major factor in spreading of infection
• ·         Fomites do not transmit the disease
• ·         The only non-venereal infection is Ophthalmia neonatorum/conjunctivitis of the newborn.- once very common, now controlled by the practice of administering 1% silver nitrate solution into the eyes of all newborns

 Mode of transmission

1.      Sexual transmission: Acquired during unprotected sex with infected partner.

2.      Neonates acquire Neisseria gonorrhoeae from mother during passage through the birth canal.  In newborn infants, Neisseria gonorrhoeae causes Ophthalmia neonatorum (purulent conjunctivitis).

(contd..)

Microbial Transport - Transport of nutrients by bacteria

The first step in nutrient use is uptake of the required nutrients by the microbial cell. Uptake mechanisms must be specific. The necessary substances alone and not others, must be acquired because a cell need not take in a substance that it cannot use. 

Microorganisms often live in nutrient-poor habitats, so they must be able to transport nutrients from dilute solutions into the cell against a concentration gradient. Also, nutrient molecules must pass through a selectively permeable plasma membrane that prevents the free passage of most substances.

In view of the enormous variety of nutrients and the complexity of the task, it is well-known that microorganisms make use of several different transport mechanisms - passive, active and group translocation, symport, antiport and uniport, electrogenic and electro neutral transport, transport of Iron.

The most important of these are facilitated diffusion, active transport, and group translocation.

Passive Diffusion

A few substances, such as glycerol, can cross the plasma membrane by passive diffusion. Passive diffusion, often called diffusion or simple diffusion, is the process in which molecules move from a region of higher concentration to one of lower concentration. The rate of passive diffusion is dependent on the size of the concentration gradient between a cell’s exterior and its interior. These do not require energy input.

A fairly large concentration gradient is required for adequate nutrient uptake by passive diffusion (i.e., the external nutrient concentration must be high while the internal concentration is low). The rate of uptake decreases as more nutrient is acquired unless it is used immediately. Very small molecules such as H₂O, O₂, and CO₂ often move across membranes by passive diffusion. Larger molecules, ions, and polar substances must enter the cell by other mechanisms.

Facilitated Diffusion

The rate of diffusion across selectively permeable membranes is greatly increased by using carrier proteins, sometimes called permeases, which are embedded in the plasma membrane. Diffusion involving carrier proteins is called facilitated diffusion. The rate of facilitated diffusion increases with the concentration gradient much more rapidly and at lower concentrations of the diffusing molecule than that of passive diffusion.

Some permeases are related to the major intrinsic protein (MIP) family of proteins. MIPs facilitate diffusion of small polar molecules. They are observed in almost all organisms. The two most widespread MIP channels in bacteria are aquaporins, which transport water and the glycerol facilitators, which aid glycerol diffusion.

Once the carrier is saturated The diffusion rate reaches a plateau because the carrier protein is binding and transporting as many solute molecules as possible. The resulting curve resembles an enzyme-substrate curve and is different from the linear response seen with passive diffusion.

Carrier proteins also resemble enzymes in their specificity for the substance to be transported; each carrier is selective and will transport only closely related solutes.

 Although a carrier protein is involved, facilitated diffusion is truly diffusion.

• ·       A concentration gradient spanning the membrane drives the movement of molecules
• ·       No metabolic energy input is required.

If the concentration gradient disappears, net inward movement ceases. The gradient can be maintained by transforming the transported nutrient to another compound.

Simple Diffusion & Facilitated Diffusion

The mechanism of facilitated diffusion is not yet understood completely. The carrier protein complex spans the membrane. After the solute molecule binds to the outside, the carrier may change conformation and release the molecule on the cell interior. The carrier subsequently changes back to its original shape and is ready to pick up another molecule.

                            

The net effect is that a hydrophilic molecule can enter the cell in response to its concentration gradient. The mechanism is driven by concentration gradients and therefore is reversible. If the solute’s concentration is greater inside the cell, it will move outward. Because the cell metabolizes nutrients upon entry, influx is favoured.

Although glycerol is transported by facilitated diffusion in many, facilitated diffusion does not seem to be the major uptake mechanism. This is because nutrient concentrations often are lower outside the cell.

Facilitated diffusion is much more prominent in eucaryotic cells where it is used to transport a variety of sugars and amino acids.

Facilitated diffusion can efficiently move molecules to the interior only when the solute concentration is higher on the outside of the cell. Microbes must have transport mechanisms that can move solutes against a concentration gradient because microorganisms often live-in habitats characterized by very dilute nutrient sources.

Microbes use two important transport processes in such situations: active transport and group translocation. Both are energy-dependent processes.

(contd..)