DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC.
Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.
Barnes
M H
MH
Department of Pharmacology, University of Massachusetts Medical School, Worcester 01655.
Tarantino
P M
PM
Spacciapoli
P
P
Brown
N C
NC
Yu
H
H
Dybvig
K
K
D000595:Amino Acid Sequence; D000963:Antimetabolites / Q000494:pharmacology; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014498:Uracil / Q000031:analogs & derivatives / Q000494:pharmacology
D000963:Antimetabolites; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C011284:6-(4-hydroxyphenylhydrazino)uracil; D014498:Uracil; D004258:DNA Polymerase III