Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima.
An expressed sequence tag homologous to cheA was previously isolated by 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 Mg(2+)-dependent autophosphorylation with [32P]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.
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
R V
RV
Swanson
M G
MG
Sanna
M I
MI
Simon
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002633:Chemotaxis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D008274:Magnesium; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010755:Organophosphates; D011993:Recombinant Fusion Proteins; C000607606:cheY protein, E coli; C011632:acetyl phosphate; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D008274:Magnesium