User:Kinkreet/MCBII/Prokaryotic

Signal Transduction
Bacterial signalling network monitors changes in the extracellular and intracellular environment, and will respond to any changes in these environments or to unfavourable environments. When a change is detected, the response produced can be rapid or slow, short-term or long-term, specific or global. Signal transduction, by definition, requires at least two components - one to detect the signal and at least one to mediate that signal to an effect. In the vast majority of cases, the signal binds to the N-terminus of a membrane receptor, this causes autophosphorylation at the C-terminal domain. The phosphate at the C-terminal domain then can transfer itself to a receiver molecule and activates/inhibits it, producing an effect such as increased or reduced gene transcription; or it can act as a docking site for other proteins, which may be scaffold proteins. Most signal transduction pathways contain more than two components, these tend to be more sensitive because the signalling cascade usually amplifies the signal.

Needless to say, the range of mechanism for detection, transduction and effect enforcement is diverse, but by comparing the genes for different molecules involved in the pathways, one can identify general trends in all these molecules. For example, most sensor molecules detect the signal at the N-terminal domain, and the signal is transmitted to the C-terminal domain (N-to-C flow). The sensory domain used can be shared between different receptors, common ones include the PAS or GAF domains; likewise, one type of stimulus can activate several pathways. Most uses secondary messenger such as cAMP and cyclic diguanylate. Different signal transduction pathways share common components, and so there are crosstalks between the different pathways.

Comparative trends were also established. Even taking into account the small genome size, parasitic bacteria encode fewer signalling proteins than free-living bacteria; Gram+ bacteria and archaea have fewer signal transduction proteins than proteobacteria or cyanobacteria which have very similar genome size.

Benefits of genomic analysis of signal transduction
When the genomes of various bacteria were sequenced, they were compared to identify the common signal transduction proteins in all organisms, as well as identify gene products specific to the species.

Histidine kinases Cyclic AMP, adenylate cyclase - chemotaxis Phosphotransferase Chemotaxis

methyl-carrier proteins or methyl-accepting chemotaxis protein (MCP) - chemotaxis by transmitting the signal to the flagellar motor and also affect transcription. diguanylate cyclase - produces c-di-GMP - involved in producing biofilm, which keeps the bacteria slimy; as well as motility and development phosphodiesterase serine/threonine protein kineases and phosphatases

Domains
Response regulators - the molecules which mediates the response from the initial detection to the effector - typically contain a phosphate-accepting receiver domain, CheY (the chemotaxis transducer protein also has the same name, but here we refer to the domain), or a DNA-binding helix-turn-helix (HTH) domain, winged helix, SAPR, LytTR, Fis or domains of other families. These domains are shared widely between different response regulators and also to other otherwise unrelated proteins.

Some domains, such as the citrate or nitrate binding domains, have high specificity towards its ligands; while others, such as the PAS domain, have low specificity and will bind any flat heterocyclic molecules from haeme to flavin to cinnamic acid and maybe the adenine moiety of ATP.

Sensory domains
Usually ligand-binding

Signal transduction
Phosphorylation, methylation, homodimerization

Output
DNA-binding, heterodimerization or enzymatic

Diffusion
The rate of diffusion is proportional to the electrochemical potential, which can be expressed as


 * $$\bar{\mu}_i=RT \ln{\frac{C_2}{C_1}} + z_iF\Phi$$,

where:
 * $$\bar{\mu}_i$$ is the electrochemical potential of species i, J/mol
 * $$R$$ is the gas constant, J K−1 mol−1
 * $$T$$ is the temperature, K
 * $$C_2$$ is the concentration of the molecule in the compartment the molecule is flowing into, M
 * $$C_2$$ is the concentration of the molecule in the compartment the molecule is flowing out from, M
 * $$\mu_i$$ is the chemical potential of the species i, J/mol
 * $$z_i$$ is the valency (charge) of the ion i, dimensionless
 * $$F$$ is Faraday's Constant, C/mol
 * $$\Phi$$ is the local electrostatic potential, V.

The equation can be split into two parts, with $$RT \ln{\frac{C_2}{C_1}}$$ representing the chemical potential, and $$z_iF\Phi$$ representing the electrical potential. Therefore, the electrochemical potential is simply a sum of the chemical and electrical potential.

Proton motive force
The proton motive force (PMF) can be seen as a derivative from the electrochemical force, where it is the sum of the proton potential and the electric potential.

Plasma Membrane
Both the Gram+ and Gram- bacteria plasmam membrane contains a cytoplasmic membrane, consisting of phospholipids and membrane proteins. This is followed by a periplasmic space and a peptidoglycan layer. These three features are common in both types of bacteria. The Gram- bacteria have an extra periplasmic space and outer membrane beyond the peptidoglycan layer.

Gram+
The peptidoglycan layer of Gram+ is thick, and contains many peptide crossbridges holding the different sub-layers of the peptidoglycan together. The surface of Gram+ bacteria expresses different markers than those of Gram- bacteria. There are general markers for Gram+ bacteria such as lipoteichoic acid (LTA), but also more specific markers for a species of Gram+ bacteria - Streptococcus expresses the M protein.

Gram-
In Gram- bacteria, the peptidoglycan layer is relatively thinner. It also has an outer membrane which consists of lipopolysaccharides (LPS). The structure of LPS consists of three parts, the O-antigen, the core and lipid A. O-antigen is the glycan polymer part of LPS which makes up the outermost part of the outer membrane; the presence of absence of the O-antigen determines whether a bacteria appear smooth or rough. This also means that bacteria with rough surfaces are more prone to hydrophobic antibiotics, because they interact more favourably with the hydrophobic O-antigens. The O-antigen is attached to the core, which can be further split into the outer and inner core, which serves to link the O-antigen to lipid A. Lipid A is typically a phosphorylated glucosamine disaccharide with many fatty acids, which provides it with the hydrophobicity that allows it to anchor into the outer membrane. Lipid A is also the toxic component of Gram- bacteria; when the immune system breaks down the bacteria, if fragments of lipid A is released into the circulation, it can cause fever, diarrhea, and possible fatal endotoxic shock (also called septic shock).

Transport
Substances need to be transported in and out of the bacteria cell, some simply move through porins and channels down their electrochemical gradient, while others require active transport in and out of the cell. Usually, the active transport system in Gram- bacteria is more sophisticated than in Gram+ bacteria, simply due to the fact that it has two membranes rather than one.

There are, however, some transporters which are globally observed in all phyla, such as the ATP-binding cassette transporters (ABC-transporter), defined by its sequence homology to the ABC domain. The ABC transport a huge range of molecules, and utilizes ATP for energy.

Porins
Porins are very common transporters on the outer membranes of Gram- bacteria; different porins have different specificities and allows different moeities through. The largest proteins that can pass through a proin is 500-700 Da, anything larger than that needs to be broken down by enzymes before being transported in. There may be a selective filter which increases the specificity of the molecules allowed through, over and above the size of the molecule.

FepA
FepA is an integral bacterial outer membrane porin protein, which is involved in the active transport of iron from the extracellular space, into the periplasm ofGram-negative bacteria. A Gram-negative bacterium secretes iron-binding proteins called siderophores, which bind strongly to ferric ions. The FepA receptor is present at the outer membrane and binds to ferric siderophores, and actively transport it into the cell. FepA has also been shown to transportvitamin B12, and colicins B and D as well.

Because no energy is directly available to the outer membrane, FepA has been studied to determine the mechanism by which energy is brought to the outer membrane to drive the transport. It was found that the energy to drive the active transport originates from the proton motive force (electrochemical gradient) generated in the inner membrane complex TonB–ExbB–ExbD, and this force is relayed physically to FepA through the TonB subunit.

Structure


Using X-ray crystallography the structure of FepA was found to be a 724-residue 22-stranded β-barrel. The barrel contains loops that act as high-affinity and high-specificity ligand-binding sites outside the cell. The N-terminus forms a smaller barrel domain inside the hydrophilic barrel pore, effectively closing the pore; from studies of FhuA, a similar TonB-dependent outer membrane transporter, the interaction of the N-terminus domain to the inner walls of the pore is thought to be strengthened by nine salt-bridges and over 60 hydrogen bonds. The N-terminus also has a further two extracellular loops in the pore, which are thought to aid in the signal transduction between ligand-binding and TonB-mediated transport, though the precise mechanism is not clear. Residues 12 to 18 of the N-terminus domain of FepA comprises a region called the TonB box, which includes at least a proline and glycine residue. The TonB box interacts with a proline-rich motif consisting of a conserved Gln160 residue and the C-terminus 48 residues. When they interact, the conformation of the N-terminal domain is changed so as to open the pore. In vivo crosslinking confirms that this interaction is physical. It is however energetically nonsensical to remove the whole of the N-terminal domain for translocation, because this requires the breakage of the salt bridges and numerous hydrogen bonds, and so it is assumed that the displace is only slight, just large enough for transport of FeEnt. The role of the N-terminus is revealed by using a deletion mutation of the N-terminal plug; the protein was still able to be inserted into the membrane, but acts as a non-selective pore for larger molecules, exhibited by increased permeability of the cell to maltotetraose, maltopentaose,ferrichrome, as well as several antibiotics including albomycin, vancomycin andbacitracin. However, this have to be treated with caution, as the conformation of the barrel may change in the absence of the N-terminal plug.

Enterobactin is a cyclic tri-ester of 2,3-dihydroxybenzoylserine with a molecular mass of 719 Da. It binds ferric ions using six oxygens from threecatechol groups, giving an overall charge of −3. Like the binding catechol, enterobactin is thought to also have a three-fold symmetry dissecting the metal centre.

Function
Iron is not usually readily available in the environment this group of bacteria find themselves in. However iron is essential in sustaining life due to its role in co-enzymes of respiration and DNA synthesis, so bacteria must adapt to have a mechanism for intake of iron. Because Fe3+ has a very low solubility, most of the Fe3+ ions in the bacteria’s surrounding environment (e.g. soil) exist as iron oxides or hydroxides, and so the number of free Fe3+ is low. Therefore, microbes have evolved to secret siderophores, Fe3+-binding peptides, into the surroundings and then actively transport the Fe3+-complex back into the cell byactive transport. This can also be seen with pathogenic bacteria inside its host, where iron is bound tightly byhaemoglobin, transferrin, lactoferrin and ferritin, and thus low in concentration (10−24 mol L−1). Here it secrets siderophores which has a higher affinity (with a formation constant, or ([ML])/([M][L]), of 1049)to Fe3+ than the host's iron-binding proteins, and so will remove iron and then transported inside the cell. Bacillus anthracis, a Gram-negative bacteria that causes anthrax, secretes two siderophores:bacillibactin and petrobactin. Escherichia coli secrets many iron-siderophore transports, but produce only one siderophore—enterobactin. The ferric enterobactin receptor FepA recognises the catecholate part of ferric enterobactin (FeEnt), and transports it across the outer membrane from the extracellular space into the periplasm. The binding is thought to be in two phases, a fast step which recognises FeEnt, and a slower step which may be the first step in translocation—preparing the complex for translocation. Both steps occur independently of the TonB–ExbB–ExbD complex and the proton motive force it provides. In the periplasm, FeEnt is bound by FepB and passed to the integral inner membrane proteins FepG and FepD through active transport, with the energy provided by ATP hydrolysis catalysed by cytoplasmic FepC. In the cytoplasm, the Fes enterobactin esterase hydrolyses and this cleaves enterobactin, releasing Fe3+ which will subsequently be reduced by the same protein, Fes, to Fe2+.

Possible Mechanisms
The 14P of the TonB box is essential for interaction, as its mutation to isoleucine led to null interaction; from this, it was also suggested that the interaction is conformational, and not sequence-specific. As there are a limited number of the TonB–ExbB–ExbD complex, it has been shown that it interacts more favourably with FepA that has a ligand bound to it. The mechanism of transport has been described as similar to a air lock. When the ligand is bound, it closes the pore at the extracellular side, and thus preventing anything from exiting through the pore. FepA then interacts with TonB, which induces a conformational change to the N-terminal, and so open the pore at the periplasmic side. This would allow FepA to transport FeEnt without allowing ions and small molecules from passing in either direction, which would occur if the system contains only a single plug.

Secretion
There are known to be at least 6 modes of secretion in Gram- bacteria. Type II secretion involves three protein subunits: the ABC protein, membrane fusion protein (MFP), and outer membrane protein (OMP); they form a single pore from the cytoplasm to the extracellular space. Type II secretion occurs as a two-staged process - it depends on the Sec or Tat pathway for translocation across the inner membrane, and uses pore forming secretin proteins to translocate across the outer membrane.

Type II secretion
A protein with an appropriate N-terminal signal sequence are transported to the Sec complex, at which point it will be transported across the inner membrane. In the periplasmic space, the signal peptide is cleaved and chaperon proteins aid the peptide to fold into the correct conformation. The outer membrane secretin would recognize the tertiary structure of the folded protein and transport it across the outer membrane. Xcp Q in P. aeroginosa is a widely studied and well characterized secretin pore. Because the outer membrane cannot generate energy it self, Xcp Q must interact with Xcp R and Xcp S of the inner membrane for them to provide the energy required for transport.

Phosphate transferase systems
The phosphate transferase system (PTS) is an important system in bacteria, it phosphorylates sugars that enter the bacterial cell, such as glucose, fructose, mannose, mannitol and cellobiose. This gives them a negative charge which prevents them from leaving the cell. The phosphate which gets added onto the sugars are ultimately derived from phosphoenolpyruvate (PEP). The sugars can be transported in using different transporters and channels.

Catabolite repression
Catabolite repression, or more specifically carbon catabolite repression, is an adaptation system that bacteria has which allows them to metabolite the most preferred choice of energy source first; for example metabolizing glucose is more efficient than metabolizing lactose, so in the presence of glucose and lactose, the bacteria would spend all its available machinary to produce proteins which take in glucose, and will inhibit the intake of lactose by not producing proteins which takes in lactose, and often inhibiting the intake.

Flagellum
There are two types of flagella, those that twist and those that rotate. In bacteria, all flagella are the rotating type.

Most of the flagella is made up of flagellin, which arranges itself to form a hollow cylinder. The N- and C-terminus of flagellin forms the core of the flagellin, and it is these domains which allows it to polymerize. The structure of flagellin contributes to the helical structure of the flagellum. At the base of the flagellum, where it is anchored to the cell, there is a hook - a sharp bend which directs the tip of the flagellum away from the axis of the bacteria.

The energy of rotation is provided by the proton motive force, which is cultivated by the Mot complex. It takes typically 1000 protons to pass through to provide enough energy for one rotation. They typical rate of rotation is more than 100 revolutions per second.

The flagella is known to be used for locomotion, for the bacteria to move towards more favourable conditions, such as a place with more nutrients. Because its length is so short, it would not be possible to accurately measure differences in concentrations of gradient across the bacteria; to move towards a source of nutrient, it uses a tumble-and-run mechanism, which uses temporal measurements instead of spatial ones. The bacteria would move forward in one direction, by turning its flagellum in an anti-clockwise direction (which causes all the flagella to align in one direction), and if after a certain time, it senses the concentration of nutrients have increased, it will continue in this direction; if, however, it senses a lowering in concentration, the flagellum will rotate clockwise and this causes the flagella to point in different directions, causing it to stop and tumble, gaining a new direction to travel to, and begin to run again. In the absence of nutrients, it would tumble every few seconds, but less tumbling occurs in high-nutrient-environments. For this to be possible, the bacteria must have some sort of molecular memory. This is also called a random walk, because the direction to which the bacteria travels after tumbling, is in practice, random. This effect has been observed in Salmonella typhimurium.

The motor can rotate in both directions. Anti-clockwise (forward) is the null condition, and tumbling requires a signal. CheY is one of proteins (CheY is also the domain which CheY the protein has) that transduct signals from membrane-bound histidine kinases. Phosphorylated CheY signals to the motor to rotate clockwise. In the absence of an attractant, such as serine, aspartate, maltose, dipeptides, galactose, ribose and oxygen, the receptor will oligomerize closely, bringing CheW and CheA domains together; the CheA domains autophosphorylate each other and the phosphate is then transferred to CheY, which signal will be transduced and causes the motor to rotate clockwise. As soon as a signal is present, the CheZ protein dephosphorylates CheY so it does not tumble indefinitely. In the presence of an attractant, the aspartate and glutamate residues repel each other and so autophosphorylation of CheA is not observed.

The molecular memory which allows the bacteria to know whether it is moving towards a more favourable environment or not is provided by methylation. In the absence of a signal, acidic residues get methylated by CheR and so neutralises the charges, allowing the different subunits of the receptor to come together, where CheA can autophosphorylate each other. CheA also activates CheB, a protein which counter the effect of CheR by demthylating the residues.