When the bacterium is exposed to a chemotactic molecule it effectively makes a choice between which mode of flagellar rotation it will use, depending upon the type of chemotactic molecule encountered. In the presence of a chemoattractant, the bacterium spends a greater proportion of its motile time with its flagella rotating in the anti-clockwise fashion. This results in the bacterium exhibiting the tumbling motion less frequently, and consequently travelling in a straight line for longer. Hence, the bacterium can target the source of the chemoattractant, and move towards it. Conversely, in the presence of a chemorepellent, the bacterium will exhibit tumbling motion much more frequently, by rotating its flagella in a clockwise fashion for longer. Hence, the presence of the chemorepellent will cause the bacterium to undergo many changes in the direction of its movement, and this will assist in evading contact with the detected chemorepellent.
Both forms of behaviour shown in the presence of chemotactic molecules show a time dependency of occurrence which is directly related to the concentration of the chemotactic molecule to which the bacterium is exposed. However, there is another aspect to the behaviour of a bacterial cell in the presence of a chemoattractant. When the bacterium encounters nutrient molecules, it begins to travel in a straight line for longer, in an attempt to find the source of this chemoattractant, and hence to expose itself to the maximum concentration possible of nutrients. As the bacterium gets closer to the source, it will spend less time displaying this chemotactic response and will eventually return to the tumbling motion. This process is called Adaptation and is the mechanism whereby bacteria become desensitised to chemotactic stimuli. This is an important aspect of the chemotactic response, allowing the bacterium to localise itself at the source of the chemoattractant.
Adaptation occurs via a complex mechanism involving a number of specialised bacterial proteins. It is triggered by prolonged exposure to a specific chemoattractant, and ensures that the bacterium does not ' overshoot ' the source of the chemoattractant which it is seeking. Adaptation is specific for a given chemoattractant, and if the adapted bacterium is then exposed to a new chemoattractant, the observed tumbling motion is abolished, and the bacterium begins to travel for longer periods of time in a straight line - towards the source of the new chemoattractant.
To understand the bacterial chemotactic mechanism, great use has been made of a range of mutants with specific chemotactic deficiencies. The classical scientific approach of isolating mutants in particular cellular processes to elucidate the mode of action of wild type individuals has a number of important aspects. Firstly, by isolating mutants in the process of interest - chemotaxis in this case - from the wild type forms of the organism, genetic components active in the phenotype of interest can be identified. Secondly, the mutant genome may then be compared to that of the wild type so that differences at the genetic level can be studied. Finally, correlation of the findings with databases on the genome of the organism of interest can then be used to allow understanding of the function of that region of the genome.
Studies on these mutants have contributed to the present understanding of the bacterial chemotactic mechanism by allowing the components of the process to be investigated. The crucial components, it seems, are the periplasmic receptor proteins, the MCP's, and the bacterial flagellum. Together, they facilitate the process of Signal Transduction Chemotaxis.
Signal Transduction Chemotaxis is the name given to the chemotactic mechanism exhibited by the gram negative bacteria. It involves a signalling cascade in which the presence of external chemotactic molecules is sensed by the bacterium and this presence is converted into a signal to stimulate movement. The movement which results is either towards the chemotactic molecule or away from it, and the chemotactant may be at a very small concentration compared to the level of intracellular components which it ultimately mobilises. The effect is to cause various cellular components to increase in their concentrations in a defined and highly regulated order, whereby one molecule in the cascade elicits the activation of many downstream molecules.
Gram negative bacteria cells have a double membrane arrangement. The outer membrane is highly permeable to many compounds, ranging from water to sugars and dipeptides, the inner membrane is very impermeable. This arrangement essentially provides a filtering system, and is the basis of the transduction mechanism. Chemotactic molecules build up in the periplasmic space, and some are recruited by periplasmic receptor proteins to permit them to interact with the downstream MCP's.
MCP's come in four distinct types, Type I / II / III / IV. Types I and II MCP's respond to amino acids, whereby Type I responds to Serine, and Type II responds to Aspartate. Type III responds to sugars and Type IV to dipeptides. The interaction of chemotactants, which the bacterium encounters in its external environment, with the MCP's is facilitated by a number of periplasmic located proteins, called the periplasmic receptor proteins. These proteins mediate the interaction of chemotactants with the Type III and Type IV MCP's. Hence, they are concerned with the binding of sugars and dipeptides, which have entered the periplasmic space from the bacterium's external environment and their recruitment to the inner bacterial membrane. Other chemotactants, such as amino acids do not need transducing periplasmic receptor proteins to allow them to interact with the MCP's, but do so via a direct intermolecular interaction.
The chemotactants to which the bacterium responds have freely entered the periplasmic space from the external environment via simple diffusion down their respective concentration gradients. Their passage across the outer bacterial membrane is unhindered, but many accumulate in the periplasmic space, being excluded from further entry by the inner bacterial membrane. Their presence is responded to by the MCP's, which are activated by the chemotactants either directly, in the case of the Types I and II MCP's, or via the transducing periplasmic receptor proteins in the case of the Types III and IV MCP's. The interaction with the MCP's serves to alter an intracellular signalling cascade in the bacterium, the result of which is that chemoattractants elicit anti-clockwise flagellar rotation, and chemorepellents elicit clockwise flagellar rotation.
The periplasmic receptor proteins communicate the presence of chemotactic molecules in the external environment to the Methyl Accepting Chemotaxis proteins (MCP's). The MCP's then transduce these signals into an intracellular signalling cascade which stimulates the appropriate mode of flagellar rotation.
The MCP's are a family of closely related transmembrane receptor proteins. They have a homodimeric structure which has been revealed by x-ray diffraction techniques. Their three dimensional structure suggests that they are embedded in the inner membrane of the bacterium, with a periplasmic - located binding domain and intracellular coiled - coil domains. The periplasmic domains of the MCP's serve to bind chemotactic molecules either directly or via the mediation of a periplasmic receptor protein. The intracellular coiled - coil domains contain the methylation sites which are vital in the process of adaptation. There are four methylation sites on each of the two polypeptide chains which constitute the homodimeric MCP. Additionally, each of the two polypeptide chains has a signalling domain, which is vital in the transduction process.
The transduction of the presence of the chemotactic molecule into an intracellular effect is a key stage in the process. Through gene studies, it has become clear that there are four cytoplasmic proteins involved in the signalling cascade downstream of the MCP's. These proteins are CheA, CheW, CheY and CheZ. They effectively couple the MCP's to the flagellar motor, and do this by using a phosphorylation relay system.
How a bacterial cell responds to the presence of a chemorepellent is a useful example to illustrate the process of the signal transduction mechanism. Chemorepellents elicit clockwise flagellar rotation, bacterial cell ' random walks ' and increased MCP activity. The mechanism involves the range of che gene products. When a repellent binds to an MCP, the resultant increase in the activity of the MCP allows it to bind to CheW, and to CheA. CheA is a histidine protein kinase - an enzyme which, when activated, can phosphorylate histidine residues in target proteins. CheW acts as a co-activator of the activity of CheA, in conjunction with the MCP. Hence, the first step after the MCP binds a chemorepellent, is the recruitment of the CheA / CheW proteins to the MCP and the activation of the histidine protein kinase activity of CheA by the action of the activated MCP and CheW.
Subsequently, CheA autophosphorylates itself upon a specific histidine residue and almost instantly transfers this high-energy phosphate to CheY. This transfer step maintains the energy of the high-energy phosphate, and generates CheY-phosphate. CheY-phosphate is the activated form of CheY, and it then binds to the flagellar motor, causing it to rotate in a clockwise fashion, causing the chaotic, tumbling movement of the bacterial cell, whereby the bacterium embarks upon a 'random walk'. CheZ, a protein phosphatase, then dephosphorylates the CheY-phosphate to inactivate it. Whether or not the flagellum then continues to spin in a clockwise fashion depends upon whether the level of CheY-phosphate is replenished, which in turn depends upon whether the chemorepellent persists.
Chemoattractants have the opposite effects. Chemoattractants binding to the MCP's decrease the activity of the MCP's. This prevents the recruitment of the CheW and the CheA proteins to the MCP's, hence reducing the phosphorylation of the CheA. This in turn reduces the phosphorylation of CheY, resulting in the level of inactivated CheY increasing. With no activated CheY, there is no mechanism to induce clockwise rotation of the bacterial flagellum. Hence, the flagellum spins in a anti-clockwise fashion, resulting in smooth swimming.
As each of the phosphorylated components of the pathway has a lifetime of approximately ten seconds, and the phosphorylation reactions occur so fast that the bacterium can be seen to have effected a chemotactic response within two hundred milliseconds of the detection of a chemotactant in its external environment. Hence, the method which is employed by bacteria serves as a fast and highly regulated response. As soon as the bacterium's environment is changed it can almost instantly respond to these changes in a rapid and appropriate way.
Adaptation allows the bacterium to efficiently position itself at the source of a chemoattractant. The effect of adaptation is achieved via methylation of the MCP at the intracellular methylation sites located upon its coiled - coil domains. There are four potential methylation sites on each of the two polypeptide chains of the MCP. Hence, there are eight potential sites for methylation upon the MCP's.
Using S-adenosylmethionine, the bacterium methylates these sites, via the mediation of a methyl transferase enzyme. The methylation can be reversed by the action of a methyl esterase enzyme when the adaptation response is no longer required ( this is a hydrolytic reaction ). The methyl groups are added to the Carboxyl groups of specific Glutamate residues on the MCP's – at the eight specific sites for methylation
The methylation process restores the normal tumbling frequency of the bacterium, and results in the bacterium displaying the chaotic movements. The methylation simply restores the activity of the MCP to its former level, meaning that the level of MCP activity is the same in the resting state and the adapted state. Thus, the bacterium can efficiently localise itself at the sources of a chemoattractant, and make the maximum use of this desirable commodity.
