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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]. The flagella are 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. The membrane anchor also functions as a rotary engine, driven by the proton motive force [4], which can either rotate clockwise or counter-clockwise. 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.

Different taxis pathways that steer cells towards favorable conditions and away from danger work by regulating the frequency of tumbling events [5], increasing tumbling in unfavorable environments and decreased the tumbling rate under favorable conditions. This form of movement, combining tumbling and running, with regulation of the tumbling frequency is termed a biased random walk [5].

At the molecular level 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].

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