Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria.
Sequences of the recA genes of the highly divergent thermopholic eubacteria Thermus aquaticus (and Thermus thermophilus), Thermotoga maritima, and Aquifex pyrophilus were determined from fragments derived by polymerase chain reaction (PCR) with degenerate primers and from inverse PCR products obtained using unique primers based on the fragment sequences. The source of the PCR products was verified by Southern hybridization. Complete PCR-derived recA genes were cloned into an expression vector regulated by a temperature-sensitive lambda-repressor, and independently derived clones expressing thermostable recA were selected. DNA sequences were verified to be authentic by direct cycle-sequencing of PCR products and/or sequencing of several clones. In contrast to Escherichia coli RecA protein, all the purified thermophilic RecA proteins exhibited single-stranded DNA-dependent ATPase activity optima above 70 degrees C. Phylogenetic analysis of RecA sequences suggested that the thermophilic RecA proteins were at least as different from one another as were Gram-positive organisms, mesophilic Gram-negative organisms, and cyanobacteria. In spite of substantial sequence divergence, interesting characteristics of the thermostable RecA proteins included increased valine content, common amino acid replacements at two highly conserved sites, and an increase in the calculated isoelectric point of approximately a full pH unit.
Wetmur
J G
JG
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029.
Wong
D M
DM
Ortiz
B
B
Tong
J
J
Reichert
F
F
Gelfand
D H
DH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D005798:Genes, Bacterial / Q000235:genetics*; D014644:Genetic Variation; D006088:Gram-Negative Aerobic Bacteria / Q000145:classification / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000145:classification / Q000235:genetics*; D006358:Hot Temperature; D006868:Hydrolysis; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000235:genetics*; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000235:genetics
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases