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Cloning and Functional Characterization of an NAD + -Dependent DNA Ligase from Staphylococcus aureus

Authors :
Dongxu Sun
Patrick K. Martin
Melissa Cronan
Bret Benton
Matt Griffor
Ajith V. Kamath
Thomas D. Gootz
Donald P. Biek
Mahmoud N. Mansour
Laura McDowell
John P. Mueller
Richard P. Zaniewski
Dennis E. Danley
Molly B. Schmid
Frank S. Kaczmarek
Source :
Journal of Bacteriology. 183:3016-3024
Publication Year :
2001
Publisher :
American Society for Microbiology, 2001.

Abstract

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus lig A gene. Orthologues of the putative S. aureus NAD + -dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli , and the enzyme was purified to near homogeneity. NAD + -dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32 P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD + -dependent DNA ligase from B. stearothermophilus , two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Details

ISSN :
10985530 and 00219193
Volume :
183
Database :
OpenAIRE
Journal :
Journal of Bacteriology
Accession number :
edsair.doi.dedup.....b5bc90111aa0159c0182f1967b57c0be
Full Text :
https://doi.org/10.1128/jb.183.10.3016-3024.2001