Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima

An expressed sequence tag homologous to cheA was previously isolated hy random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR, Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced, The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function, Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors, Upon induction, both proteins were synthesized at high levels, T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min, CheY prepared in this way was shown to be active by the demonstration of Mg2+-dependent autophosphorylation with [P-32]acetyl phosphate, In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.