Part:BBa_K343003

NpSopII-NpHtrII-StTar (M-fusion)

Sensory Rhodopsin II bluelight receptor fused to its transducer, HtrII, with a 27 BP linker region. This is fused to Salmonella enterica serovar typhimurium chemotaxis protein Tar, so that the protein effectively couples the input from the receptor to the chemotaxis pathway and reduces the amount of phosphorylated CheY, which results in a lowered tumbling frequency. The fusion between HtrII and Tsr is an M-fusion in the HAMP domain, which is supposed to give it maximum activity. Sequencing confirmed that the sequence is identical to the found in the article by Jung, Spudich E, Trivedi and Spudich J in the article: An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli. (1)

Background

The mechanism of bacterial motility

Bacterias main form of propulsion in liquids is thorugh swimming with help of flagella. A single flagellum is a thin filament around 100-150 Å thick, that extends many cell lengths out from the cell [2]. It consists mainly of flagellin subunits that assemble into a helical structure forming a long hollow cylindrical filament [3]. The environment around the cell also has a large influence on how many flagellae are present, or if they are present at all [4].

A flagellum is anchored to the cell body by a large, wheel-like protein complex spanning both inner and outer membranes [4]. Through this complex subunits are secreted to the tip of the flagellar tube[3], thus elongating the filament (This mechanism is slightly different in archaea, where the filament is assembled from the base of the flagellum). The membrane anchor also functions as a rotary engine, driven by the proton motive force [4]. In E. coli the flagellar motor rotates at up to 300hz and can turn either clockwise or counter-clockwise [4], both resulting in a distinct movement pattern for the cell.

Spinning in the counter-clockwise direction, the flagella will twist into a bundle in the shape of a corkscrew, and create a linear driving force [4], propelling the cell in a straight line through the liquid. This form of movement is termed run. Spun in the clockwise direction one might then expect the cell to reverse, but this is not the case. Instead the flagellar bundle will unwind, thereby creating chaotic movement [4] . This movement reorients the cell randomly and is termed tumbling. A cell will typically run for some time, then change it’s orientation by tumbling, and then run again [5]. The direction of flagella rotation is controlled by the binding of a cytosolic protein CheY, more on which later [4].

Different taxis pathways that steer cells towards favorable conditions and away from danger work by regulating the frequency of tumbling events [5]. An example is when a bacterium gets close to the source of a lethal toxin, then intracellular pathways will increase the frequency of tumbling events, in effect preventing the cell from dying. Since the frequency of tumbling events will decrease if the cell is going in a direction away from the toxin, it will “encourage” the cell to continue in that direction. In the case of an attractant such as an increase in nutrient concentration, the pattern will be opposite, so that the cell is encouraged to continue towards the source of the attractant. This form of movement, combining tumbling and running, with regulation of the tumbling frequency is termed a biased random walk [5].

To understand how this can work we need a simplified understanding of the chemotaxis pathway at a molecular level. Chemotactic receptors can both increase and decrease tumbling frequencies to generate a biased random walk behavior[6]. Increased tumbling is achieved through a phosphorylation cascade beginning with the binding of a repellant to a transmembrane receptor[4]. The receptor is linked to two proteins CheW and CheA[4]. CheA is a histidine-kinase that will autophosphorylate when the repellant binds[4]. The phosphoryl group is then transferred to CheY[4]. The flagellar motor complex has high affinity for phosphorylated CheY (CheY-p), and binding reverses the mode of movement from run to tumble[7]. CheY-p is continuously dephosphorylated back to CheY by CheZ[8]. A receptor sensing an attractant might instead switch from the default active CheA state to an inactive state when it’s ligand is bound, thus decreasing CheY phosphorylation [6].

Molecular mechanism of the photosensor

The fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2)

As described above modification of taxis happens through modification of the bacterial tumbling frequency. The photosensor acts directly on the E. Coli's chemotaxis pathway, which we outlined in the paragraph above. The Sensory Rhodopsin II, is fused to the N-terminal portion of the transducer protein HtrII. This in turn is again fused to the cytoplasmic portion of the salmonellar chemotaxis receptor Tar (2), where the HAMP domains of the two proteins are located, to achieve a uniform response in the complex. It is the Tar protein that binds to CheW and CheA, which are part of E. Colis normal chemotaxis pathway. When exposed to bluelight, the sensory rhodopsin II will absorb the photons and therefore tilt one of its transmembrane helices outwards (3), which changes the ultrastructure of the linked HtrII protein, which also passes the signaling on to Tar. This leads to a lower rate ofautophosphorylation of CheA, which in turn again decreases the amount of phosphorylated CheY. The site of this response is dependent on the amount of methylation of the protein CheA This means that the amount of phosphorylated CheY will be smaller than normally in E.Coli. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. How the sensory rhodopsin II acts on the bacteria depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepair of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1783
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 73
  Illegal NgoMIV site found at 331
  Illegal AgeI site found at 1585
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1027
  Illegal BsaI.rc site found at 1300
  Illegal SapI site found at 801
  Illegal SapI.rc site found at 1801